Stevia plant having less ability to form flower buds

ABSTRACT

The present invention provides a  stevia  plant having a low ability to form flower buds as compared with the wild type  stevia  species. The present invention also provides a method of producing such a  stevia  plant having a low ability to form flower buds, and an extract or a steviol glycoside purified product obtainable from such a plant.

TECHNICAL FIELD

The present invention relates to a stevia plant having a low ability toform flower buds, a method for producing the same and a method forscreening for the same, etc.

BACKGROUND ART

Stevia is a perennial plant of the family Asteraceae with Paraguay inthe South America as its place of origin. Stevia contains a sweetcomponent having several tens to several hundreds of times the sweetnessof sugar, and such a sweet component is extracted therefrom and used asa natural sweetener (Patent Literature 1). However, much remains unknownabout gene information or what kind of gene is involved in the controlof in vivo events in stevia, for example.

CITATION LIST Patent Literature

-   Patent Literature 1: WO2018/124142

SUMMARY OF INVENTION Technical Problem

It is desired to further elucidate gene information on stevia.

Means for Solving the Problems

The present invention provides a stevia plant having a low ability toform flower buds, a method of producing the plant, and a method ofscreening for the plant, etc.

In one embodiment, the present invention provides the following.

[1] A stevia plant having a low ability to form flower buds as comparedwith the wild type.[2] The plant according to [1], wherein the plant is heterozygous orhomozygous for the allele wherein the base at the position correspondingto position 90 of SEQ ID NO: 150 is G, and/or is heterozygous orhomozygous for the allele wherein the base at the position correspondingto position 108 of SEQ ID NO: 150 is G.[3] The plant according to [1] or [2], further having at least one ofthe following genetic features (1) to (7).

(1) Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T.

(2) Homozygous for the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T.

(3) Homozygous for the allele wherein the base at the positioncorresponding to position 41 of

SEQ ID NO: 4 is C.

(4) Homozygous for the allele wherein the portion corresponding topositions 55-72 of SEQ ID NO: 5 is deleted.

(5) Homozygous for the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A.

(6) Heterozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A.

(7) Homozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A.

[4] The plant according to [3], having at least one of the followingfeatures (1) and (2).

(1) Comprising 3% or more of RebD per unit mass of a dried leaf.

(2) Comprising 0.2% or more of RebM per unit mass of a dried leaf.

[5] The plant according to any one of [1] to [4], wherein the plant is anon-genetically modified plant.[6] The plant according to any one of [1] to [5], wherein the plantcomprises a stevia plant subjected to a mutagenesis treatment and aprogeny plant thereof.[7] A seed, a tissue, a tissue culture or a cell of the plant accordingto any one of [1] to [6].[8] The tissue, tissue culture or cell according to [7], which isselected from an embryo, a meristem cell, a pollen, a leaf, a root, aroot apex, a petal, a protoplast, a leaf section and a callus.[9] A method of producing a stevia plant having a low ability to formflower buds, the method comprising a step of crossing the plantaccording to any one of [1] to [6] with a second stevia plant.[10] The method according to [9], wherein the second plant is the plantaccording to any one of [1] to [6].[11] An extract of the plant according to any one of [1] to [6], or ofthe seed, tissue, tissue culture or cell according to [7] or [8].[12] A method of producing a stevia extract, comprising a step ofobtaining an extract from the plant according to any one of [1] to [6],or from the seed, tissue, tissue culture or cell according to [7] or[8].[13] A method of producing a steviol glycoside purified product,comprising: a step of obtaining an extract from the plant according toany one of [1] to [6], or from the seed, tissue, tissue culture or cellaccording to [7] or [8]; and a step of purifying a steviol glycosidefrom the obtained extract.[14] The method according to [13], wherein the steviol glycosidecomprises rebaudioside A, rebaudioside B, rebaudioside C, rebaudiosideD, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N,rebaudioside 0, stevioside, steviolbioside, rubusoside, dulcoside A or acombination thereof.[15] A method of producing a food or beverage, a sweetener composition,a flavor or a medicament, comprising:

a step of providing the extract according to [11], or a purified productthereof; and

a step of adding the extract or the purified product to a raw materialfor the food or beverage, sweetener composition, flavor or medicament.

[16] A method of screening for a stevia plant with a low ability to formflower buds, comprising a step of detecting from the genome of a teststevia plant the presence and/or the absence of a genetic feature ofbeing heterozygous or homozygous for the allele wherein the base at theposition corresponding to position 90 of SEQ ID NO: 150 is G, and/or thebase at the position corresponding to position 108 of SEQ ID NO: 150 isG.[17] The method according to [16], further comprising a step ofdetecting from the genome of a test stevia plant the presence and/or theabsence of the following genetic features (1) to (7).

(1) Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T.

(2) Homozygous for the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T.

(3) Homozygous for the allele wherein the base at the positioncorresponding to position 41 of SEQ ID NO: 4 is C.

(4) Homozygous for the allele wherein the portion corresponding topositions 55-72 of SEQ ID NO: 5 is deleted.

(5) Homozygous for the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A.

(6) Heterozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A from the genome of thetest stevia plant.

(7) Homozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A from the genome of thetest stevia plant.

[18] The method according to [16] or [17], wherein the step of detectinga genetic feature is performed by use of CAPS method, dCAPS method orTaqMan PCR method.[19] The method according to any one of [16] to [18], further comprisinga step of evaluating the ability to form flower buds in a test steviaplant tissue.[20] A screening kit for a stevia plant with a low ability to formflower buds, comprising a reagent for detecting the presence and/or theabsence of a genetic feature of being heterozygous or homozygous for theallele wherein the base at the position corresponding to position 90 ofSEQ ID NO: 150 is G, and/or being heterozygous or homozygous for theallele wherein the base at the position corresponding to position 108 ofSEQ ID NO: 150 is G.[21] The kit according to [20], further comprising a reagent fordetecting the presence and/or the absence of the following geneticfeatures (1) to (7).

(1) Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T.

(2) Homozygous for the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T.

(3) Homozygous for the allele wherein the base at the positioncorresponding to position 41 of SEQ ID NO: 4 is C.

(4) Homozygous for the allele wherein the portion corresponding topositions 55-72 of SEQ ID NO: 5 is deleted.

(5) Homozygous for the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A.

(6) Heterozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A.

(7) Homozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A.

[22] The kit according to [20] or [21], wherein the reagent comprises aprimer and/or a probe for use in CAPS method, dCAPS method or TaqMan PCRmethod.[23] A method of producing a stevia plant with a low ability to formflower buds, comprising a step of introducing a variation from A to G toa position corresponding to position 90 of SEQ ID NO: 150, and/or a stepof introducing a variation from T to G to a position corresponding toposition 108 of SEQ ID NO: 150.[24] The method according to [23], wherein the introduction of thevariation is performed by a mutagenesis treatment.

Advantageous Effects of Invention

The present invention enables the obtainment of a stevia plant having alow ability to form flower buds and the provision of an approach forproducing such a plant, a leaf obtainable from such a plant, and a food,a drink, etc. containing an extract obtained from this leaf. When theability to form flower buds is low, nutrients that are supposed to beused for flower bud formation are used in leaf growth. Thus, improvementin leaf productivity and by extension, increase in contents of steviolglycosides accumulated in leaves can be expected.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing a frequency distribution of sweet componentcontents in M1 generation individuals. The ordinate depicts the numberof individuals, and the abscissa depicts a sweet component concentration(%) in a dried leaf.

FIG. 2 is a diagram showing a distribution of sweet component contentsin variation C49A positive individuals (C49A⁺) and negative individuals(C49A⁻) of segregating population A. The ordinate depicts a sweetcomponent concentration (%) in a dried leaf, and the dotted line depictsan average sweet component concentration of all the individualsbelonging to segregating population A.

FIG. 3 is a diagram showing a distribution of sweet component contentsin variation C49A positive individuals (C49A⁺) and negative individuals(C49A⁻) of segregating population B. The ordinate depicts a sweetcomponent concentration (%) in a dried leaf, and the dotted line depictsan average sweet component concentration of all the individualsbelonging to segregating population B.

DESCRIPTION OF EMBODIMENTS

The present invention will now be described in detail. The followingembodiments are provided for illustrating the present invention and arenot intended to limit the present invention only thereto. The presentinvention may be implemented in various forms, without departing fromthe spirit of the present invention.

Note that all documents, as well as laid-open application publications,patent application publications, and other patent documents cited hereinshall be incorporated herein by reference. The present specificationincorporates the contents of the specification and the drawings ofJapanese Patent Application No. 2019-075610, filed on Apr. 11, 2019,from which the present application claims priority.

1. Stevia Plant Having a Low Ability to Form Flower Buds

The present invention provides a stevia plant having a low ability toform flower buds as compared with the wild type (hereinafter,generically referred to as the “plant of the present invention” or“stevia plant of the present invention”).

Stevia is a plant having a scientific name of Stevia rebaudiana Bertoni.

The phrase “having a low ability to form flower buds as compared withthe wild type” means that the number of flower buds formed for apredetermined period is smaller than that of the wild type stevia plantwhen the plants to be compared are cultivated, for example, undershort-day conditions and under the same cultivation conditions. Morespecifically, the phrase means that the number of flower buds formed fora predetermined period is smaller by 20% or more, 25% or more, 30% ormore, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more,60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 95% or more, or 100% than that of the wild typestevia plant when the plants to be compared are cultivated, for example,under short-day conditions and under the same cultivation conditions.The phrase “smaller by 100%” means that no flower bud is formed (i.e.,the number of flower buds is 0) while the wild type stevia plant has oneor more formed flower buds. The short-day conditions involve a darkphase of longer than 10 hours, preferably 11 hours or longer. Thepredetermined period means a period for which the wild type stevia plantforms flower buds under short-day conditions, and may be, for example, 2weeks, 3 weeks, or 4 weeks. Also, 5 or less, for example, 5, 4, 3, 2, 1or 0 flower buds after cultivation for 3 weeks under conditions of a12-hour light phases, or 10 or less, for example, 10, 9, 8, 7, 6, 5, 4,3, 2, 1 or 0 flower buds after cultivation for 3 weeks under conditionsof an 8-hour light phases may be used as an alternative index for thephrase “having a low ability to form flower buds as compared with thewild type”.

In one embodiment, the stevia plant of the present invention has atleast one genetic feature (hereinafter, referred to as the “geneticfeature X of the present invention”) selected from a genetic feature ofbeing homozygous or heterozygous for the allele wherein the base at theposition corresponding to position 90 of SEQ ID NO: 150 is G(hereinafter, referred to as the “genetic feature X-1 of the presentinvention”) and a genetic feature of being homozygous or heterozygousfor the allele wherein the base at the position corresponding toposition 108 of SEQ ID NO: 150 is G (hereinafter, referred to as the“genetic feature X-2 of the present invention”).

In one embodiment, the stevia plant of the present invention has agenetic feature of being homozygous for the allele wherein the base atthe position corresponding to position 201 of SEQ ID NO: 1 is A(hereinafter, referred to as the “genetic feature A of the presentinvention”).

In another embodiment, the stevia plant of the present invention has atleast one of the following genetic features (B-1) to (B-4) (hereinafter,referred to as the “genetic feature B of the present invention”).

(B-1) Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T (hereinafter, referredto as the “genetic feature B-1 of the present invention”).

(B-2) Homozygous for the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T (hereinafter, referredto as the “genetic feature B-2 of the present invention”).

(B-3) Homozygous for the allele wherein the base at the positioncorresponding to position 41 of SEQ ID NO: 4 is C (hereinafter, referredto as the “genetic feature B-3 of the present invention”).

(B-4) Homozygous for the allele wherein the portion corresponding topositions 55-72 of SEQ ID NO: 5 is deleted (hereinafter, referred to asthe “genetic feature B-4 of the present invention”).

In an alternative embodiment, the stevia plant of the present inventionhas a genetic feature of being heterozygous for the allele wherein thebase at the position corresponding to position 49 of SEQ ID NO: 6 is A(hereinafter, referred to as the “genetic feature C of the presentinvention”).

In an alternative embodiment, the stevia plant of the present inventionhas a genetic feature of being homozygous for the allele wherein thebase at the position corresponding to position 49 of SEQ ID NO: 6 is A(hereinafter, referred to as the “genetic feature D of the presentinvention”).

In a preferable embodiment, the stevia plant of the present inventionhas the genetic feature X (i.e., at least one of the genetic featuresX-1 and X-2 of the present invention) and the genetic feature A of thepresent invention. In another preferable embodiment, the stevia plant ofthe present invention has the genetic feature X and the genetic featureB (i.e., at least one of the genetic features B-1 to B-4 of the presentinvention) of the present invention. In an alternative preferableembodiment, the stevia plant of the present invention has the geneticfeature X and the genetic feature C or D of the present invention. In analternative preferable embodiment, the stevia plant of the presentinvention has the genetic feature X, the genetic feature A, and thegenetic feature B of the present invention. In an alternative preferableembodiment, the stevia plant of the present invention has, the geneticfeature X, the genetic feature A, and the genetic feature C or D of thepresent invention. In an alternative preferable embodiment, the steviaplant of the present invention has the genetic feature X, the geneticfeature B, and the genetic feature C or D of the present invention. In amore preferable embodiment, the stevia plant of the present inventionhas all of the genetic features X, A, B and C or D of the presentinvention.

The phrase “position (or portion) corresponding to” means the following.In case a sequence identical to a reference sequence (e.g., SEQ ID NOs:1 to 6, 150 etc.) is present in the genome, it means a position or aportion in the sequence (e.g., 201, 40, 44, 41, 55-72, 49, 90/108, etc.)present in the genome, and in case a sequence identical to the referencesequence is not present in the genome, it means a position or portion ina sequence in the genome corresponding to the reference sequence, whichcorresponds to the position or portion in the reference sequence.Whether or not a sequence identical to or corresponding to the referencesequence exists in the genome can be determined by, for example,amplifying genomic DNA of the stevia plant of interest with a primercapable of amplifying the reference sequence by PCR, sequencing theamplified product, and performing alignment analysis between theobtained sequence and the reference sequence. Non-limiting examples of asequence corresponding to a reference sequence include, for example, anucleotide sequence having a sequence identity of 60% or more, 70% ormore, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more,84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4%or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or99.8% or more to the reference sequence. The position or portioncorresponding to the position or portion in the reference sequence inthe sequence corresponding to the reference sequence in the genome canbe determined by taking into account the nucleotide sequence before andafter the position or portion in the reference sequence and the like.For example, a position or portion in the sequence corresponding to thereference sequence in the genome corresponding to a position or portionin the reference sequence can be determined by an alignment analysis ofa reference sequence with a sequence corresponding to a referencesequence in the genome.

For instance, when taking “the position corresponding to position 201 ofSEQ ID NO: 1” of the genetic feature A of the present invention as anexample, in case the genome of a stevia plant has a portion consistingof a nucleotide sequence identical to SEQ ID NO: 1, “the positioncorresponding to position 201 of SEQ ID NO: 1” is position 201 from the5′ end of the portion consisting of a nucleotide sequence identical toSEQ ID NO: 1 in the genome. On the other hand, in case the genome of astevia plant has a portion consisting of a nucleotide sequence which isnot identical to, but which corresponds to SEQ ID NO: 1, the genome doesnot have a portion consisting of a nucleotide sequence identical to SEQID NO: 1. Therefore, “the position corresponding to position 201 of SEQID NO: 1” does not necessarily correspond to position 201 from the 5′end of the portion corresponding to SEQ ID NO: 1. However, it ispossible to identify “the position corresponding to position 201 of SEQID NO: 1” in the genome of such a stevia plant by taking into accountthe nucleotide sequence before and after the position 201 of SEQ ID NO:1, and the like. For instance, one can identify “the positioncorresponding to position 201 of SEQ ID NO: 1” in the genome of a steviaplant by an alignment analysis of the nucleotide sequence of a portioncorresponding to SEQ ID NO: 1″ in the genome of a stevia plant and thenucleotide sequence of SEQ ID NO: 1.

“The portion consisting of a nucleotide sequence corresponding to SEQ IDNO: 1” means, for instance, a portion consisting of a nucleotidesequence having a sequence identity of 60% or more, 70% or more, 75% ormore, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more,85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% ormore, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more,96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more,98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% ormore to the nucleotide sequence of SEQ ID NO: 1.

In one embodiment, “the portion consisting of a nucleotide sequencecorresponding to SEQ ID NO: 1” includes a portion of the genome of astevia plant which can be amplified by PCR using a forward primer whichhybridizes to a complementary sequence of a portion of 15 to 25 baselong from the 5′ end of SEQ ID NO: 1 and a reverse primer whichhybridizes to a portion of 15 to 25 base long from the 3′ end of SEQ IDNO: 1.

For simplicity, the genetic feature A of the present invention is usedhere as an example for explanation, but the same applies to the geneticfeatures X (including genetic features X-1 and X-2), B (including thegenetic features B-1 to B-4), C and D of the present invention.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 150” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:151 and a reverse primer comprising the nucleotide sequence of SEQ IDNO: 152.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 1” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:7 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:8.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 2” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:9 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:10.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 3” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:11 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:12.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 4” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:13 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:14.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 5” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:15 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:16.

In a specific embodiment, “the portion consisting of a nucleotidesequence corresponding to SEQ ID NO: 6” includes, for instance, aportion of the genome of a stevia plant which can be amplified by PCRusing a forward primer comprising the nucleotide sequence of SEQ ID NO:17 and a reverse primer comprising the nucleotide sequence of SEQ ID NO:18.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position 90 of SEQ ID NO: 150 is G” comprises thenucleotide sequence of SEQ ID NO: 153 or 154. In a specific embodiment,“the allele wherein the base at the position corresponding to position108 of SEQ ID NO: 150 is G” comprises the nucleotide sequence of SEQ IDNO: 155 or 156.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A” comprises thenucleotide sequence of SEQ ID NO: 19, 20 or 21.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position of SEQ ID NO: 2 is T” comprises the nucleotidesequence of SEQ ID NO: 22, 23 or 24.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T” comprises thenucleotide sequence of SEQ ID NO: 25, 26 or 27.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position 41 of SEQ ID NO: 4 is C” comprises thenucleotide sequence of SEQ ID NO: 28, 29 or 30.

In a specific embodiment, “the allele wherein the portion correspondingto positions 55-72 of SEQ ID NO: 5 is deleted” comprises the nucleotidesequence of SEQ ID NO: 31, 32 or 33.

In a specific embodiment, “the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A” comprises thenucleotide sequence of SEQ ID NO: 34, 35 or 36.

Here, a position selected from the group consisting of (X-1) a positioncorresponding to position 90 of SEQ ID NO: 150, (X-2) a positioncorresponding to position 108 of SEQ ID NO: 150, (A) a positioncorresponding to position 201 of SEQ ID NO: 1, (B-1) a positioncorresponding to position 40 of SEQ ID NO: 2, (B-2) a positioncorresponding to position 44 of SEQ ID NO: 3, (B-3) a positioncorresponding to position 41 of SEQ ID NO: 4, (B-4) a portioncorresponding to positions 55-72 of SEQ ID NO: 5, and (C) a positioncorresponding to position 49 of SEQ ID NO: 6 may be generically referredto as a “polymorphic site of the present invention” or a “variation siteof the present invention”.

Also, a variation selected from the group consisting of (X-1) avariation from A to G at a position corresponding to position 90 of SEQID NO: 150, (X-2) a variation from T to G at a position corresponding toposition 108 of SEQ ID NO: 150, (A) a variation from C to A at aposition corresponding to position 201 of SEQ ID NO: 1, (B-1) avariation from A to T at a position corresponding to position 40 of SEQID NO: 2, (B-2) a variation from C to T at a position corresponding toposition 44 of SEQ ID NO: 3, (B-3) a variation from G to C at a positioncorresponding to position 41 of SEQ ID NO: 4, (B-4) a deletion of theportion corresponding to positions 55-72 of SEQ ID NO: 5, and (C) avariation from C to A at a position corresponding to position 49 of SEQID NO: 6 may be generically referred to as a “polymorphism of thepresent invention” or a “variation of the present invention”.

The above genetic features can be detected by PCR method, TaqMan PCRmethod, sequencing method, microarray method, Invader method, TILLINGmethod, RAD (random amplified polymorphic DNA) method, restrictionfragment length polymorphism (RFLP) method, PCR-SSCP method, AFLP(amplified fragment length polymorphism) method, SSLP (simple sequencelength polymorphism) method, CAPS (cleaved amplified polymorphicsequence) method, dCAPS (derived cleaved amplified polymorphic sequence)method, allele-specific oligonucleotide (ASO) method, ARMS method,denaturing gradient gel electrophoresis (DGGE) method, CCM (chemicalcleavage of mismatch) method, DOL method, MALDI-TOF/MS method, TDImethod, padlock probe method, molecular beacon method, DASH (dynamicallele specific hybridization) method, UCAN method, ECA method, PINPOINTmethod, PROBE (primer oligo base extension) method, VSET (very shortextension) method, Survivor assay, Sniper assay, Luminex assay, GOODmethod, LCx method, SNaPshot method, Mass ARRAY method, pyrosequencingmethod, SNP-IT method, melting curve analysis method, etc., butdetection methods are not limited thereto.

In a specific embodiment, each genetic feature of the present inventionis detectable using the following combination of a primer set and arestriction enzyme.

In case a candidate plant has the genetic feature X-1, for example,bands of approximately 90 bp long (e.g., SEQ ID NO: 160) andapproximately 25 bp (e.g., SEQ ID NO: 161) are obtained by: performingPCR amplification using a forward primer having the nucleotide sequenceshown in SEQ ID NO: 157 and a reverse primer having the nucleotidesequence shown in SEQ ID NO: 158 on the genomic DNA of the candidateplant; and treating the obtained PCR product (approximately 115 bp long,e.g., SEQ ID NO: 158) with a restriction enzyme AluI. On the other hand,when only a band of approximately 115 bp long (e.g., SEQ ID NO: 162) isobtained by: obtaining, for example, a PCR product (approximately 115 bplong) of SEQ ID NO: 162 by PCR amplification; and treating this PCRproduct with a restriction enzyme AluI, the candidate plant does nothave the genetic feature X-1.

In case a candidate plant has the genetic feature X-2, for example,bands of approximately 112 bp long (e.g., SEQ ID NO: 165) andapproximately 25 bp (e.g., SEQ ID NO: 166) are obtained by: performingPCR amplification using a forward primer having the nucleotide sequenceshown in SEQ ID NO: 157 and a reverse primer having the nucleotidesequence shown in SEQ ID NO: 163 on the genomic DNA of the candidateplant; and treating the obtained PCR product (approximately 137 bp long,e.g., SEQ ID NO: 164) with a restriction enzyme ApaI. On the other hand,when only a band of approximately 137 bp long (e.g., SEQ ID NO: 167) isobtained by: obtaining, for example, a PCR product (approximately 137 bplong) of SEQ ID NO: 167 by PCR amplification; and treating this PCRproduct with a restriction enzyme ApaI, the candidate plant does nothave the genetic feature X-2.

In case a candidate plant has the genetic feature A, for example, a bandof approximately 96 bp long (e.g., SEQ ID NO: 41) and a band ofapproximately 100 bp (e.g., SEQ ID NO: 42) are obtained by: performingPCR amplification using a forward primer having the nucleotide sequenceshown in SEQ ID NO: 37 and a reverse primer having the nucleotidesequence shown in SEQ ID NO: 38 on the genomic DNA of the candidateplant; and treating the obtained PCR product (approximately 196 bp long,e.g., SEQ ID NO: 39) with a restriction enzyme Hpy188I. On the otherhand, when restriction enzyme-treated products of approximately 43 bp(e.g., SEQ ID NO: 43) and approximately 57 bp (e.g., SEQ ID NO: 44) areformed by: obtaining, for example, a PCR product (approximately 196 bplong) of SEQ ID NO: 40 by PCR amplification; and treating the PCRproduct with a restriction enzyme Hpy188I, the candidate plant does nothave the genetic feature A.

In case where a candidate plant has the genetic feature B-1, forexample, only a band of approximately 297 bp long (e.g., SEQ ID NO: 47)is obtained by: performing PCR amplification using a forward primerhaving the nucleotide sequence shown in SEQ ID NO: 45 and a reverseprimer having the nucleotide sequence shown in SEQ ID NO: 46 on thegenomic DNA of the candidate plant; and treating the obtained PCRproduct (approximately 297 bp long: e.g., SEQ ID NO: 47) with a KpnIrestriction enzyme. On the other hand, when a restriction enzyme-treatedproduct of approximately 258 bp (e.g., SEQ ID NO: 49) is formed by:obtaining, for example, a PCR product (approximately 297 bp long) of SEQID NO: 48 by PCR amplification; and treating the PCR product with arestriction enzyme KpnI, the candidate plant does not have the geneticfeature B-1.

In case where a candidate plant has the genetic feature B-2, forexample, only a band of approximately 383 bp long (e.g., SEQ ID NO: 52)is obtained by: performing PCR amplification using a forward primerhaving the nucleotide sequence shown in SEQ ID NO: 50 and a reverseprimer having the nucleotide sequence shown in SEQ ID NO: 51 on thegenomic DNA of the candidate plant; and treating the obtained PCRproduct (approximately 383 bp long: e.g., SEQ ID NO: 52) with an XbaIrestriction enzyme. On the other hand, when a restriction enzyme-treatedproduct of approximately 344 bp long (e.g., SEQ ID NO: 54) is formed by:obtaining, for example, a PCR product (approximately 297 bp long) of SEQID NO: 53 by PCR amplification; and treating the PCR product with arestriction enzyme XbaI, the candidate plant does not have the geneticfeature B-2.

In case where a candidate plant has the genetic feature B-3, forexample, only a band of approximately 390 bp long (e.g., SEQ ID NO: 57)is obtained by: performing PCR amplification using a forward primerhaving the nucleotide sequence shown in SEQ ID NO: 55 and a reverseprimer having the nucleotide sequence shown in SEQ ID NO: 56 on thegenomic DNA of the candidate plant; and treating the obtained PCRproduct (approximately 390 bp long: e.g., SEQ ID NO: 57) with an AflIIrestriction enzyme. On the other hand, when a restriction enzyme-treatedproduct of approximately 347 bp long (e.g., SEQ ID NO: 59) is formed by:obtaining, for example, a PCR product (approximately 297 bp long) of SEQID NO: 58 by PCR amplification; and treating the PCR product with arestriction enzyme AflII, the candidate plant does not have the geneticfeature B-3.

In case where a candidate plant has the genetic feature B-4, forexample, only a PCR product of approximately 140 bp (e.g., SEQ ID NO:62) is formed by performing PCR amplification using a forward primerhaving the nucleotide sequence shown in SEQ ID NO: 60 and a reverseprimer having the nucleotide sequence shown in SEQ ID NO: 61 on thegenomic DNA of the candidate plant. On the other hand, when PCR productsof 140 bp long (e.g., SEQ ID NO: 62) and 158 bp long (e.g., SEQ ID NO:63) are formed, the candidate plant does not have the genetic featureB-4.

In case a candidate plant has the genetic feature C or D, for example, aband of approximately 367 bp long (e.g., SEQ ID NO: 66) and a band ofapproximately 321 bp (e.g., SEQ ID NO: 68) are obtained by: performingPCR amplification using a forward primer having the nucleotide sequenceshown in SEQ ID NO: 64 and a reverse primer having the nucleotidesequence shown in SEQ ID NO: 65 on the genomic DNA of the candidateplant; and treating the obtained PCR product (approximately 367 bp long:e.g., SEQ ID NO: 66) with a restriction enzyme SpeI. On the other hand,when only a restriction enzyme-treated product of approximately 367 bplong (e.g., SEQ ID NO: 67) is formed by: obtaining, for example, a PCRproduct (approximately 367 bp long) of SEQ ID NO: 67 by PCRamplification; and treating the PCR product with a restriction enzymeSpeI, the candidate plant has neither of genetic feature C nor D.

The term “approximately” as to bp long described above means±5 bp. Therestriction enzyme treatment can be performed according to conditionsrecommended by the distributor of each restriction enzyme used.

The ability to form flower buds can be evaluated by, for example, anyknown approach or an approach described in Example 6. Non-limitingexamples of the method of evaluating the ability to form flower budsinclude the following approach:

(1) A test stevia plant is cultivated for a predetermined period undershort-day conditions.(2) The number of formed flower buds is counted.

The test stevia plant may be cultivated alone or may be cultivatedtogether with the wild type stevia plant (control) in the sameenvironment. In the case of cultivating the test stevia plant alone, theevaluation approach described above may comprise step of comparing thenumber of flower buds formed in the test stevia plant with the number offlower buds formed in the wild type stevia plant cultivated undersimilar conditions (e.g., based on data obtained in a literature or aseparate experiment). In the case of cultivating the test stevia planttogether with the wild type stevia plant, the evaluation approachdescribed above may comprise a step of comparing the number of flowerbuds formed in the test stevia plant with the number of flower budsformed in the wild type stevia plant cultivated together therewith.

The short-day conditions involve, as mentioned above, a dark phase setto longer than 10 hours, preferably 11 hours or longer. A specificlength of the dark phase may be, for example, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours. Thepredetermined cultivation period is not particularly limited as long asthe wild type stevia plant is capable of forming flower buds undershort-day conditions for the period. The cultivation period may be, forexample, 2 weeks, 3 weeks, or 4 weeks.

In one embodiment, the plant of the present invention comprises 3% ormore of RebD per unit mass of a dried leaf. This means that, forexample, RebD is contained at a ratio of 3% by mass or more (e.g., 1.5mg or more) in a dried leaf having a predetermined mass (e.g., 50 mg).In this embodiment, the ratio of RebD per unit mass of a dried leaf isnot limited and may be, for example, 3.0% or more, 3.1% or more, 3.2% ormore, 3.3% or more, 3.4% or more, 3.5% or more, 3.6% or more, 3.7% ormore, 3.8% or more, 3.9% or more, 4.0% or more, 4.1% or more, 4.2% ormore, 4.3% or more, 4.4% or more, 4.5% or more, 4.6% or more, 4.7% ormore, 4.8% or more, 4.9% or more, 5.0% or more, 5.1% or more, 5.2% ormore, 5.3% or more, 5.4% or more, 5.5% or more, 5.6% or more, 5.7% ormore, 5.8% or more, 5.9% or more, 6.0% or more or the like, and ispreferably 3.3% or more, more preferably 3.6% or more. The upper limitof the ratio of RebD per unit mass of a dried leaf is not particularlylimited and may be, for example, 20%, 15% or 10%. As shown in Examples,the genetic features A to D are highly related to this embodiment.

In this context, the dried leaf refers to a leaf having a water contentdecreased to 3 to 4% by weight by drying a fresh leaf of the steviaplant of the present invention.

In one embodiment, the plant of the present invention comprises 2.6% ormore of RebD and 0.4% or more of RebM per unit mass of a dried leaf.This means that, for example, RebD and RebM are contained at ratios of2.6% by mass or more (e.g., 1.3 mg or more per 50 mg of a dried leaf)and 0.4% by mass or more (e.g., 0.2 mg or more per 50 mg of a driedleaf), respectively, in a dried leaf having a predetermined mass (e.g.,50 mg). In this embodiment, the ratios of RebD and RebM per unit mass ofa dried leaf are not limited and may be, for example, (2.6% or more:0.4%or more), (2.8% or more:0.4% or more), (3% or more:0.4% or more), (3.2%or more:0.4% or more), (3.4% or more:0.4% or more), (3.6% or more:0.4%or more), (3.8% or more:0.4% or more), (4% or more:0.4% or more), (4.2%or more:0.4% or more), (4.4% or more:0.4% or more), (4.6% or more:0.4%or more), (4.8% or more:0.4% or more), (5% or more:0.4% or more), (2.6%or more:0.5% or more), (2.8% or more:0.5% or more), (3% or more:0.5% ormore), (3.2% or more:0.5% or more), (3.4% or more: 0.5% or more), (3.6%or more:0.5% or more), (3.8% or more:0.5% or more), (4% or more:0.5% ormore), (4.2% or more:0.5% or more), (4.4% or more:0.5% or more), (4.6%or more:0.5% or more), (4.8% or more:0.5% or more), (5% or more:0.5% ormore), (2.6% or more:0.6% or more), (2.8% or more:0.6% or more), (3% ormore:0.6% or more), (3.2% or more:0.6% or more), (3.4% or more: 0.6% ormore), (3.6% or more:0.6% or more), (3.8% or more:0.6% or more), (4% ormore:0.6% or more), (4.2% or more:0.6% or more), (4.4% or more:0.6% ormore), (4.6% or more:0.6% or more), (4.8% or more:0.6% or more), (5% ormore:0.6% or more), (2.6% or more:0.7% or more), (2.8% or more:0.7% ormore), (3% or more:0.7% or more), (3.2% or more:0.7% or more), (3.4% ormore: 0.7% or more), (3.6% or more:0.7% or more), (3.8% or more:0.7% ormore), (4% or more:0.7% or more), (4.2% or more:0.7% or more), (4.4% ormore:0.7% or more), (4.6% or more:0.7% or more), (4.8% or more:0.7% ormore), (5% or more:0.7% or more), (2.6% or more:0.8% or more), (2.8% ormore:0.8% or more), (3% or more:0.8% or more), (3.2% or more:0.8% ormore), (3.4% or more: 0.8% or more), (3.6% or more:0.8% or more), (3.8%or more:0.8% or more), (4% or more:0.8% or more), (4.2% or more:0.8% ormore), (4.4% or more:0.8% or more), (4.6% or more:0.8% or more), (4.8%or more:0.8% or more), (5% or more:0.8% or more) or the like in terms of(ratio of RebD:ratio of RebM), and are preferably (3.6% or more:0.4% ormore). The upper limit of the ratio of RebD per unit mass of a driedleaf is not particularly limited and may be, for example, 20%, 15% or10%. Likewise, the upper limit of the ratio of RebM is not particularlylimited and may be, for example, 10%, 5% or 3%. As shown in Examples,the genetic features A to D are highly related to this embodiment.

In one embodiment, the plant of the present invention comprises 3.7% ormore in total of RebD and RebM per unit mass of a dried leaf. This meansthat, for example, the total mass of RebD and RebM contained in a driedleaf having a predetermined mass (e.g., 50 mg) is 3.7% by mass or more(e.g., 1.85 mg or more). In this embodiment, the total ratio of RebD andRebM per unit mass of a dried leaf is not limited and may be, forexample, 3.7% or more, 3.8% or more, 3.9% or more, 4.0% or more, 4.1% ormore, 4.2% or more, 4.3% or more, 4.4% or more, 4.5% or more, 4.6% ormore, 4.7% or more, 4.8% or more, 4.9% or more, 5.0% or more, 5.1% ormore, 5.2% or more, 5.3% or more, 5.4% or more, 5.5% or more, 5.6% ormore, 5.7% or more, 5.8% or more, 5.9% or more, 6.0% or more, 6.1% ormore, 6.2% or more, 6.3% or more, 6.4% or more, 6.5% or more, 6.6% ormore, 6.7% or more, 6.8% or more, 6.9% or more, 7.0% or more or thelike, and is preferably 4.9% or more. The upper limit of the total ratioof RebD and RebM per unit mass of a dried leaf is not particularlylimited and may be, for example, 25%, 20% or 15%. As shown in Examples,the genetic features A to D are highly related to this embodiment.

In one embodiment, in the plant of the present invention, the total massratio of RebD and RebM to total steviol glycoside is 37.8% or more. Thismeans that, for example, when the total mass of RebD and RebM containedin a leaf (e.g., a dried leaf or a fresh leaf) is indicated byRebD+RebM/TSG % as the ratio to the total mass of steviol glycosidesobtained from the leaf, the value of RebD+RebM/TSG is 37.8% or more. Inthis embodiment, the value of RebD+RebM/TSG is not limited and may be,for example, 37.8% or more, 37.9% or more, 38.0% or more, 38.1% or more,38.2% or more, 38.3% or more, 38.4% or more, 38.5% or more, 38.6% ormore, 38.7% or more, 38.8% or more, 38.9% or more, 39.0% or more, 39.2%or more, 39.4% or more, 39.6% or more, 39.8% or more, 40.0% or more,40.2% or more, 40.4% or more, 40.6% or more, 40.8% or more, 41.0% ormore, 41.2% or more, 41.4% or more, 41.6% or more, 41.8% or more, 42.0%or more, 42.4% or more, 42.8% or more, 43.2% or more, 43.6% or more,44.0% or more, 44.4% or more, 44.8% or more, 45.2% or more, 45.6% ormore, 46.0% or more or the like, and is preferably 38.1% or more. Theupper limit of the mass ratio of RebD+RebM to total steviol glycoside isnot particularly limited and may be, for example, 85%, 75%, 65% or 55%.As shown in Examples, the genetic features A to D are highly related tothis embodiment.

TSG is a generic name for measurable steviol glycosides and includesneither an unknown steviol glycoside nor a steviol glycoside present ata level less than the detection limit. Preferably, the total steviolglycoside is any combination of two or more members selected from thegroup consisting of RebA, RebB, RebD, RebE, RebF, Rebl, RebJ, RebK,RebM, RebN, RebO, RebQ, RebR, dulcoside A, rubusoside, steviolmonoside,steviolbioside and stevioside. In a certain embodiment, the totalsteviol glycoside may consist of, for example, RebA, RebB, RebM, RebD,RebF, RebM and stevioside. In another embodiment, the total steviolglycoside may consist of RebA, RebB, RebM, RebD, RebF, RebM, RebN, RebOand stevioside. In a specific embodiment, the total steviol glycosideconsists of RebA, RebB, RebC, RebD, RebF, RebM, RebN and RebO.

In one embodiment, the plant of the present invention comprises 0.2% ormore of RebM per unit mass of a dried leaf. This means that, forexample, the mass of RebM contained in a dried leaf having apredetermined mass (e.g., 50 mg) is 0.2% by mass or more (e.g., 0.1 mgor more). In this embodiment, the total ratio of RebD and RebM per unitmass of a dried leaf is not limited and may be, for example, 0.20% ormore, 0.25% or more, 0.30% or more, 0.35% or more, 0.40% or more, 0.45%or more, 0.50% or more, 0.55% or more, 0.60% or more, 0.65% or more,0.70% or more, 0.75% or more, 0.80% or more, 0.85% or more, 0.90% ormore, 0.95% or more, 1.00% or more, 1.05% or more, 1.10% or more, 1.15%or more, 1.20% or more, 1.25% or more, 1.30% or more, 1.35% or more,1.40% or more, 1.45% or more or the like, and is preferably 0.4% ormore. The upper limit of the ratio of RebM per unit mass of a dried leafis not particularly limited and may be, for example, 15%, 10% or 5%. Asshown in Examples, the genetic feature B is highly related to thisembodiment.

In one embodiment, in the plant of the present invention, the mass ratioof RebM to total steviol glycoside is 2% or more. This means that, forexample, when the mass of RebM contained in a leaf (e.g., a dried leafor a fresh leaf) is indicated by RebM/TSG % as the ratio to the totalmass of steviol glycosides obtained from the leaf, the value of RebM/TSGis 2% or more. In this embodiment, the value of RebM/TSG is not limitedand may be, for example, 2% or more, 2.5% or more, 3% or more, 3.5% ormore, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more,6.5% or more, 7% or more, 7.5% or more, 8% or more, 8.5% or more, 9% ormore, 9.5% or more, 10% or more, 10.5% or more, 11% or more, 12% ormore, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more,18% or more, 19% or more, 20% or more or the like, and is preferably3.5% or more. The upper limit of the mass ratio of RebM to total steviolglycoside is not particularly limited and may be, for example, 50%, 45%,40% or 35%. As shown in Examples, the genetic feature B is highlyrelated to this embodiment.

In one embodiment, the plant of the present invention, when the amount(g) of rebaudioside M contained per 100 g of a leaf (e.g., a dried leafor a fresh leaf) of the wild type stevia plant is defined as 100%,comprises rebaudioside M at higher content by 300% or more, 400% ormore, 500% or more, 600% or more, 700% or more, 800% or more, 900% ormore, 1100% or more, 1200% or more, 1300% or more, 1400% or more, 1500%or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more,2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% ormore, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900%or more, 3000% or more as compared with the wild type stevia species. Asshown in Examples, the genetic feature B is highly related to thisembodiment.

In one embodiment, the plant of the present invention comprises 1% ormore of RebD per unit mass of a dried leaf. This means that, forexample, the mass of RebD contained in a dried leaf having apredetermined mass (e.g., 50 mg) is 1% by mass or more (e.g., 0.5 mg ormore). In this embodiment, the ratio of RebD per unit mass of a driedleaf is not limited and may be, for example, 1.00% or more, 1.05% ormore, 1.10% or more, 1.15% or more, 1.20% or more, 1.25% or more, 1.30%or more, 1.35% or more, 1.40% or more, 1.45% or more, 1.50% or more,1.55% or more, 1.60% or more, 1.65% or more, 1.70% or more, 1.75% ormore, 1.80% or more, 1.85% or more, 1.90% or more, 1.95% or more, 2.00%or more, 2.05% or more, 2.10% or more, 2.15% or more, 2.20% or more,2.25% or more, 2.30% or more, 2.35% or more, 2.40% or more, 2.45% ormore, 2.50% or more, 2.55% or more, 2.60% or more, 2.65% or more, 2.70%or more, 2.75% or more, 2.80% or more, 2.85% or more, 2.90% or more,2.95% or more, 3.00% or more, 3.05% or more, 3.10% or more, 3.15% ormore, 3.20% or more, 3.25% or more, 3.30% or more, 3.35% or more, 3.40%or more, 3.45% or more, 3.50% or more, 3.55% or more, or 3.57% or moreor the like, and is preferably 0.4% or more. The upper limit of theratio of RebD per unit mass of a dried leaf is not particularly limitedand may be, for example, 15%, 10% or 5%. As shown in Examples, thegenetic feature B is highly related to this embodiment.

In one embodiment, in the plant of the present invention, when thecontent of RebM and RebD in a leaf (e.g., a dried leaf or a fresh leaf)is indicated by RebM/RebD ratio, the lower limit of the value ofRebM/RebD is 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 ormore, 0.8 or more, 1.0 or more.

On the other hand, the upper limit of the value of RebM/RebD is 0.3 orless, 0.4 or less, 0.5 or less, 0.6 or less, 0.8 or less, 1.0 or less,1.1 or less, 1.2 or less. The combination of the lower limit and theupper limit is not particularly limited as long as the upper limit valueis larger than the lower limit value in the combination. The ratio ispreferably 0.2 or more and 1.2 or less, or 0.6 or more and 1.1 or less.As shown in Examples, the genetic feature B is highly related to thisembodiment.

In one embodiment, in the plant of the present invention, when thecontent of RebM and RebD in a leaf (e.g., a dried leaf or a fresh leaf)is indicated by (RebD+RebM)/TSG % as the ratio to the total amount ofsteviol glycosides, the lower limit of the value of (RebD+RebM)/TSG is14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% ormore, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more,36% or more, 38% or more. On the other hand, the upper limit of thevalue of (RebD+RebM)/TSG is 18% or less, 20% or less, 22% or less, 24%or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% orless, 36% or less, 38% or less, 40% or less. The combination of thelower limit and the upper limit is not particularly limited as long asthe upper limit value is larger than the lower limit value in thecombination. The ratio is preferably 14% or more and 40% or less, or 16%or more and 40% or less. As shown in Examples, the genetic feature B ishighly related to this embodiment.

RebD and RebM can be extracted in the state of a liquid extract byreacting a fresh leaf or a dried leaf of the plant of the presentinvention with a suitable solvent (an aqueous solvent such as water oran organic solvent such as an alcohol, ether or acetone). For theextraction conditions, etc., see a method described in Ohta et al., J.Appl. Glycosci., Vol. 57, No. 3, 199-209 (2010) or WO2010/038911, or amethod described in Examples mentioned later.

RebD can be further purified from the liquid extract thus obtained byuse of a method known in the art such as a gradient of ethyl acetate orany of other organic solvents:water, high performance liquidchromatography (HPLC), gas chromatography, time-of-flight massspectrometry (TOF-MS), or ultra (high) performance liquid chromatography(UPLC).

The contents of RebD or RebM can be measured by a method described inOhta et al. (supra) or WO2010/038911, or a method described in Examplesmentioned later. Specifically, a fresh leaf can be sampled from thestevia plant of the present invention, followed by measurement byLC/MS-MS.

The plant of the present invention may include not only the whole plantbut a plant organ (e.g., a leaf, a petal, a stem, a root, and a seed), aplant tissue (e.g., epidermis, phloem, soft tissue, xylem, vascularbundle, palisade tissue, and spongy tissue), various forms of plantcells (e.g., suspended cultured cells), a protoplast, a leaf section, acallus, and the like. The leaf may be the dried leaf mentioned above.

The plant of the present invention may also include a tissue culture ora cultured plant cell. This is because the plant can be regenerated byculturing such a tissue culture or a cultured plant cell. Examples ofthe tissue culture or the cultured plant cell of the plant of thepresent invention include, but are not limited to, embryos, meristemcells, pollens, leaves, roots, root apices, petals, protoplasts, leafsections and calluses.

2. Method of Producing Plant of Present Invention

In an alternative aspect, the present invention provides a method ofproducing stevia plant having a lower ability to form flower buds thanthat of the wild type, the method comprising a step of crossing thestevia plant of the present invention with a second stevia plant(hereinafter, may be referred to as the “production method of thepresent invention”).

The “stevia plant having a lower ability to form flower buds than thatof the wild type” produced by the method has the same phenotype andgenetic properties as those of the plant of the present invention.

The ability to form flower buds, the ranges of the contents of RebD andRebM, etc. in the plant obtained by the production method of the presentinvention are as described above about the plant of the presentinvention.

In one embodiment, the plant obtained by the production method of thepresent invention has the genetic feature X of the present invention. Inone embodiment, the plant obtained by the production method of thepresent invention has the genetic feature A of the present invention. Inanother embodiment, the plant obtained by the production method of thepresent invention has the genetic feature B of the present invention. Inan alternative embodiment, the plant obtained by the production methodof the present invention has the genetic feature C or D of the presentinvention. In a preferable embodiment, the plant obtained by theproduction method of the present invention has the genetic feature X andthe genetic feature A of the present invention. In another preferableembodiment, the plant obtained by the production method of the presentinvention has the genetic feature X and the genetic feature B of thepresent invention. In an alternative preferable embodiment, the plantobtained by the production method of the present invention has thegenetic feature X and the genetic feature C or D of the presentinvention. In an alternative preferable embodiment, the plant obtainedby the production method of the present invention has the geneticfeature X, the genetic feature A, and the genetic feature B of thepresent invention. In an alternative preferable embodiment, the plantobtained by the production method of the present invention has thegenetic feature X, the genetic feature A, and the genetic feature C or Dof the present invention. In an alternative preferable embodiment, theplant obtained by the production method of the present invention has thegenetic feature X, the genetic feature B, and the genetic feature C or Dof the present invention. In a more preferable aspect, the plantobtained by the production method of the present invention has all ofthe genetic features X, A, B and C or D of the present invention.

In the production method of the present invention, “hybridizing” meansthat the plant of the present invention (first generation (S1)) iscrossed with a second plant (S1) to obtain a progeny plant thereof(plant produced by the production method of the present invention(second generation (S2)).

The hybridizing method is preferably backcross. The “backcross” is anapproach of further crossing a progeny plant (S2) generated between theplant of the present invention and the second plant, with the plant ofthe present invention (i.e., a plant having the genetic feature(s) ofthe present invention) (S1) to produce a plant having the geneticfeature(s) of the present invention. When the second plant (S1) for usein the production method of the present invention has the same phenotypeand genetic properties as those of the plant of the present invention,the crossing is substantially backcross. The genetic polymorphism of thepresent invention is inheritable according to the Mendel's law. Inassociation with this, the phenotype correlating with the geneticpolymorphism, i.e., the low ability to form flower buds, is alsoinheritable according to the Mendel's law.

Alternatively, the plant of the present invention can also be producedby selfing. The selfing can be performed by the self-pollination of thestamen pollen of the plant of the present invention with the pistil ofthe plant of the present invention.

Since the plant produced by the production method of the presentinvention has the same phenotype and genetic properties as those of theplant of the present invention, the plant produced by the productionmethod of the present invention can be further crossed with a thirdstevia plant to produce a stevia plant having a phenotype equivalent tothat of the plant of the present invention.

In an alternative embodiment, the plant of the present invention may beproduced by regenerating a plant by the culture of the tissue culture orthe cultured plant cell mentioned above. The culture conditions are thesame as those for culturing a tissue culture or a cultured plant cell ofthe wild type stevia plant and are known in the art (Protocols for InVitro cultures and secondary metabolite analysis of aromatic andmedicinal plants, Method in molecular biology, vo. 1391, pp 113-123).

In a further alternative embodiment, the plant of the present inventionmay be produced by introducing the variation of the present invention tothe genome of a stevia plant. The introduction of the variation may beperformed by a genetic modification approach or may be performed by anon-genetic modification approach. Examples of the “non-geneticmodification approach” include a method of inducing a variation in thegene of a host cell (or a host plant) without transfection with aforeign gene. Examples of such a method include a method of allowing amutagen to act on a plant cell. Examples of such a mutagen includeethylmethanesulfonic acid (EMS) and sodium azide. For example, theethylmethanesulfonic acid (EMS) can be used at a concentration such as0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% to treat aplant cell. The treatment time is 1 to 48 hours, 2 to 36 hours, 3 to 30hours, 4 to 28 hours, 5 to 26 hours, 6 to 24 hours. The proceduresthemselves of the treatment are known in the art and can be performed bydipping a water-absorbed seed obtained through a water absorptionprocess in a treatment solution containing the mutagen at theconcentration described above for the treatment time described above.

An alternative example of the non-genetic modification approach can be amethod of irradiating a plant cell with radiation or light beam such asX ray, y ray, or ultraviolet ray. In this case, a cell irradiated usingan appropriate dose (ultraviolet lamp intensity, distance, and time) ofultraviolet ray is cultured in a selective medium or the like, and then,a cell, a callus, or a plant having the trait of interest can beselected. In this operation, the irradiation intensity is 0.01 to 100Gr, 0.03 to 75 Gr, 0.05 to 50

Gr, 0.07 to 25 Gr, 0.09 to 20 Gr, 0.1 to 15 Gr, 0.1 to 10 Gr, 0.5 to 10Gr, 1 to 10 Gr. The irradiation distance is 1 cm to 200 m, 5 cm to 100m, 7 cm to 75 m, 9 cm to 50 m, 10 cm to 30 m, 10 cm to 20 m, 10 cm to 10m. The irradiation time is 1 minute to 2 years, 2 minutes to 1 year, 3minutes to 0.5 years, 4 minutes to 1 month, 5 minutes to 2 weeks, or 10minutes to 1 week. The irradiation intensity, distance and time differdepending on the type of radiation or the state of the subject to beirradiated (cell, callus, or plant) and can be appropriately adjusted bythose skilled in the art.

Approaches such as cell fusion, another culture (haploid induction), andremote crossing (haploid induction) are also known in the art.

In general, plant cells may involve a mutation during culture.Therefore, it is preferred to regenerate a plant individual, for morestably maintaining the trait.

The scope of the present invention does not exclude a plant obtained bythe ex-post facto genetic recombination (e.g., genome editing) with theplant of the present invention as a host (e.g., a plant further providedwith another trait by genetic recombination with the plant of thepresent invention as a host).

A plant having a plurality of different genetic features of the presentinvention may be produced by crossing plants differing from each otherin the genetic feature of the present invention. For example, a planthaving the genetic features X and A of the present invention can beobtained by crossing a plant having the genetic feature X of the presentinvention with a plant having the genetic feature A of the presentinvention. A plant having the genetic features X and B of the presentinvention, a plant having the genetic features X and C or D of thepresent invention, a plant having the genetic features A and B of thepresent invention, a plant having the genetic features A and C or D ofthe present invention, a plant having the genetic features B and C or Dof the present invention, or the like can be obtained by similarcrossing. Also, a plant having the genetic features X, A and B of thepresent invention can be obtained, for example, by crossing a planthaving the genetic features X and A of the present invention with aplant having the genetic feature B of the present invention, by crossinga plant having the genetic features X and B of the present inventionwith a plant having the genetic feature A of the present invention, orby crossing a plant having the genetic feature X of the presentinvention with a plant having the genetic features A and B of thepresent invention. A plant having the genetic features X, B and C or Dof the present invention, a plant having the genetic features A, B and Cor D of the present invention, or the like can be obtained by similarcrossing. The crossing is preferably performed over two or moregenerations. For heterozygous genetic features or the like, a planthaving the desired combination of the genetic features may be obtainedin one generation.

3. Method of Screening for Plant of Present Invention

The plant of the present invention or the plant having the samephenotype and genetic properties as those of the plant of the presentinvention can be screened for by detecting the genetic feature(s) of thepresent invention from a tissue of this plant. In this context,“screening” means that the plant of the present invention isdiscriminated from the other plants to select the plant of the presentinvention. Thus, in an alternative aspect, the present inventionprovides a method of screening for a stevia plant having a low abilityto form flower buds, comprising a step of detecting the presence and/orthe absence of at least one of the genetic features X, A, B, C and D ofthe present invention from the genome of a test plant (hereinafter, maybe referred to as the “screening method of the present invention”).

In one embodiment, the genetic feature(s) to be detected is the geneticfeature X of the present invention. In another embodiment, the geneticfeature(s) to be detected is the genetic feature A of the presentinvention. In an alternative embodiment, the genetic feature(s) to bedetected is the genetic feature B of the present invention. In analternative embodiment, the genetic feature(s) to be detected is thegenetic feature C of the present invention. In an alternativeembodiment, the genetic feature(s) to be detected is the genetic featureD of the present invention. In a preferable embodiment, the geneticfeature(s) to be detected is the genetic feature X and the geneticfeature A of the present invention. In another preferable embodiment,the genetic feature(s) to be detected is the genetic feature X and thegenetic feature B of the present invention. In an alternative preferableembodiment, the genetic feature(s) to be detected is the genetic featureX and the genetic feature C of the present invention. In an alternativepreferable embodiment, the genetic feature(s) to be detected is thegenetic feature X, the genetic feature A, and the genetic feature B ofthe present invention. In an alternative preferable embodiment, thegenetic feature(s) to be detected is the genetic feature X, the geneticfeature A, and the genetic feature C or D of the present invention. Inan alternative preferable embodiment, the genetic feature(s) to bedetected is the genetic feature X, the genetic feature B, and thegenetic feature C or D of the present invention. In a more preferableembodiment, the genetic feature(s) to be detected is all of the geneticfeatures X, A, B and C or D of the present invention.

The screening method of the present invention may further comprise astep of selecting from among the test plants a plant in which thepresence of at least one genetic feature of the above is detected.

The presence of the genetic feature(s) of the present invention can bedetermined by detecting the presence of an allele selected from thegroup consisting of:

(X-1) an allele wherein the base at the position corresponding toposition 90 of SEQ ID NO: 150 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 153 or 154);

(X-2) an allele wherein the base at the position corresponding toposition 108 of SEQ ID NO: 150 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 155 or 156);

(A) an allele wherein the base at the position corresponding to position201 of SEQ ID NO: 1 is A (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 69);

(B-1) an allele wherein the base at the position corresponding toposition 40 of SEQ ID NO: 2 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 70);

(B-2) an allele wherein the base at the position corresponding toposition 44 of SEQ ID NO: 3 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 71);

(B-3) an allele wherein the base at the position corresponding toposition 41 of SEQ ID NO: 4 is C (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 72);

(B-4) an allele wherein the portion corresponding to positions 55-72 ofSEQ ID NO: 5 is deleted (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 73); and

(C) an allele wherein the base at the position corresponding to position49 of SEQ ID NO: 6 is A (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 74); and/or

by detecting the absence of an allele selected from the group consistingof:

(x-1) an allele wherein the base at the position corresponding toposition 90 of SEQ ID NO: 150 is A (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 168 or 169);

(x-2) an allele wherein the base at the position corresponding toposition 108 of SEQ ID NO: 150 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 170 or 171);

(a) an allele wherein the base at the position corresponding to position201 of SEQ ID NO: 1 is T (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 1);

(b-1) an allele wherein the base at the position corresponding toposition 44 of SEQ ID NO: 2 is A (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 2);

(b-2) an allele wherein the base at the position corresponding toposition 40 of SEQ ID NO: 3 is C (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 3);

(b-3) an allele wherein the base at the position corresponding toposition 41 of SEQ ID NO: 4 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 4);

(b-4) an allele wherein the portion corresponding to positions 55-72 ofSEQ ID NO: 5 is not deleted (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 5); and

(c) an allele wherein the base at the position corresponding to position49 of SEQ ID NO: 6 is C (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 6).

The absence of the genetic feature(s) of the present invention can bedetermined by

detecting the absence of an allele selected from the group consistingof:

(X-1) an allele wherein the base at the position corresponding toposition 90 of SEQ ID NO: 150 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 153 or 154);

(X-2) an allele wherein the base at the position corresponding toposition 108 of SEQ ID NO: 150 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 155 or 156);

(A) an allele wherein the base at the position corresponding to position201 of SEQ ID NO: 1 is A (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 69);

(B-1) an allele wherein the base at the position corresponding toposition 40 of SEQ ID NO: 2 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 70);

(B-2) an allele wherein the base at the position corresponding toposition 44 of SEQ ID NO: 3 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 71);

(B-3) an allele wherein the base at the position corresponding toposition 41 of SEQ ID NO: 4 is C (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 72);

(B-4) an allele wherein the portion corresponding to positions 55-72 ofSEQ ID NO: 5 is deleted (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 73); and

(C) an allele wherein the base at the position corresponding to position49 of SEQ ID NO: 6 is A (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 74); and/or

by detecting the presence of an allele selected from the groupconsisting of:

(x-1) an allele wherein the base at the position corresponding toposition 90 of SEQ ID NO: 150 is A (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 168 or 169);

(x-2) an allele wherein the base at the position corresponding toposition 108 of SEQ ID NO: 150 is T (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 170 or 171);

(a) an allele wherein the base at the position corresponding to position201 of SEQ ID NO: 1 is T (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 1);

(b-1) an allele wherein the base at the position corresponding toposition 44 of SEQ ID NO: 2 is A (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 2);

(b-2) an allele wherein the base at the position corresponding toposition 40 of SEQ ID NO: 3 is C (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 3);

(b-3) an allele wherein the base at the position corresponding toposition 41 of SEQ ID NO: 4 is G (e.g., an allele comprising thenucleotide sequence of SEQ ID NO: 4);

(b-4) an allele wherein the portion corresponding to positions 55-72 ofSEQ ID NO: 5 is not deleted (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 5); and

(c) an allele wherein the base at the position corresponding to position49 of SEQ ID NO: 6 is C (e.g., an allele comprising the nucleotidesequence of SEQ ID NO: 6).

Specific examples of methods of detecting the genetic features of thepresent invention include, but not limited to, PCR method, TaqMan PCRmethod, sequencing method, microarray method, Invader method, TILLINGmethod, RAD method, RFLP method, PCR-SSCP method, AFLP method, SSLPmethod, CAPS method, dCAPS method, ASO method, ARMS method, DGGE method,CCM method, DOL method, MALDI-TOF/MS method, TDI method, padlock probemethod, molecular beacon method, DASH method, UCAN method, ECA method,PINPOINT method, PROBE method, VSET method, Survivor assay, Sniperassay, Luminex assay, GOOD method, LCx method, SNaPshot method, MassARRAY method, pyrosequencing method, SNP-IT method, melting curveanalysis method, etc.

In the case of PCR method, it is preferable to generate a primer suchthat the 3′ end portion has a sequence complementary to the polymorphicsite of the present invention. By using a primer designed in this way,the polymerase extension reaction proceeds because the primer hybridizescompletely to the template if the template sample has the polymorphism,whereas if the template does not have the variation of the presentinvention, the extension reaction does not occur because the nucleotideat the 3′ end of the primer mismatches the template. Therefore, PCRamplification is performed using such a primer, and the amplificationproduct is analyzed by agarose gel electrophoresis or the like, and ifan amplification product of a predetermined size can be confirmed, thetemplate as the sample has a variation, and if the amplification productis not present, it can be judged that the template does not have avariation.

Alternatively, the genetic feature(s) of the present invention can bedetected by designing the primer sequence so that the polymorphism ofthe present invention and the primer sequence do not overlap and thegenetic variation of the present invention can be PCR amplified, and bysequencing the nucleotide sequence of the amplified nucleotide fragment.

For PCR and agarose gel electrophoresis see Sambrook, Fritsch andManiatis, “Molecular Cloning: A Laboratory Manual” 2nd Edition (1989),Cold Spring Harbor Laboratory Press.

TaqMan PCR method uses fluorescently labeled allele-specific oligos andTaq DNA polymerases (Livak, K. J. Genet). Anal. 14, 143 (1999); MorrisT. et al., J. Clin. Microbiol. 34, 2933 (1996)).

The sequencing method is a method of analyzing the presence or absenceof a variation by amplifying a region containing the variation by PCRand sequencing the DNA sequence using a Dye Terminator or the like(Sambrook, Fritsch and Maniatis (supra)).

A DNA microarray is one in which one end of a nucleotide probe isimmobilized in an array on a support, and includes a DNA chip, a Genechip, a microchip, a bead array, and the like. By using a probecontaining a sequence complementary to the polymorphism of the presentinvention, the presence or absence of the polymorphism of the presentinvention can be comprehensively detected. DNA microarray assays such asDNA chips include GeneChip assays (see Affymetrix; U.S. Pat. Nos.6,045,996; 5,925,525; and 5,858,659). The GeneChip technique utilizes aminiaturized, high density microarray of oligonucleotide probes affixedto a chip.

The invader method combines the hybridization of two reporter probesspecific for each allele of a polymorphism such as SNPs and one invaderprobe to template DNA and the cleavage of DNA by Cleavase enzyme with aspecial endonuclease activity which cleaves a DNA by recognizing itsstructure (see, e.g., Livak, K. J. Biomol. Eng. 14, 143-149 (1999);Morris T. et al., J. Clin. Microbiol. 34, 2933 (1996); Lyamichev, V. etal., Science, 260, 778-783 (1993)).

TILLING (Targeting Induced Local Lesions IN Genomes) method is a methodin which mutational mismatches in the genomes of a mutagenized mutantpopulation are screened by PCR-amplification and CEL Inuclease-treatment.

In one embodiment, the genetic feature X-1 of the present invention canbe detected, without limitations, by dCAPS method using the followingprimer set and a restriction enzyme.

Primer set: A primer set comprising a forward primer comprising asequence (e.g., SEQ ID NO: 157) of any consecutive 15 bases or morewhich is positioned upstream of position 90 of SEQ ID NO: 150, and areverse primer comprising a sequence of any consecutive 15 to 25 baseslong from the 3′ end of a sequence selected from SEQ ID NOs: 158 and 172to 183. The sequences of the primers can be optimized within a rangethat satisfies the conditions described above. For the optimization ofprimer design, see, for example, Sambrook and Russell, “MolecularCloning: A Laboratory Manual” 3rd Edition (2001), Cold Spring HarborLaboratory Press. Each of the primers may be 15 to 50 base long, 18 to48 base long, 20 to 45 base long, 30 to 65 base long, or the like.

Restriction Enzyme:

A restriction enzyme appropriate for each of SEQ ID NOs: 158 and 172 to183 is shown below.

TABLE 1Restriction enzyme appropriate for sequence contained in reverse primerSequence contained in reverse primer Restriction enzymeTGGCGCCCCCCTGGGGCGTACACAG (SEQ ID NO: 158) AluITGGCGCCCCCCTGGGGCGTACACGG (SEQ ID NO: 172) BcefITGGCGCCCCCCTGGGGCGTACAGCG (SEQ ID NO: 173) Eco47III/HaeIITGGCGCCCCCCTGGGGCGTACGCCG (SEQ ID NO: 174) TauITGGCGCCCCCCTGGGGCGTACAGCT (SEQ ID NO: 175) AceIIITGGCGCCCCCCTGGGGCATACACTG (SEQ ID NO: 176) ApaBITGGCGCCCCCCTGGGGCGTACGCAG (SEQ ID NO: 177) BbvITGGCGCCCCCCTGGGGCCTACACGG (SEQ ID NO: 178) BglITGGCGCCCCCCTGGGGAGTACACCT (SEQ ID NO: 179) BplI/Hin4ITGGCGCCCCCCTGGGGCGTACAACA (SEQ ID NO: 180) BsbITGGCGCCCCCCTGGGGCGTACACGT (SEQ ID NO: 181) BtrITGGCGCCCCCCTGGGGCGTACGACG (SEQ ID NO: 182) HgaITGGCGCCCCCCTGGGGCGTACAAAG (SEQ ID NO: 183) HindIII

In a specific embodiment, the genetic feature X-1 of the presentinvention can be detected by dCAPS method using the following primer setand restriction enzyme.

TABLE 2 Combination of primer set and restriction enzyme Sequence ofSequence of forward primer reverse primer Restriction enzyme SEQ ID NO:157 SEQ ID NO: 158 AluI SEQ ID NO: 157 SEQ ID NO: 172 BcefI SEQ ID NO:157 SEQ ID NO: 173 Eco47III/HaeII SEQ ID NO: 157 SEQ ID NO: 174 TauI SEQID NO: 157 SEQ ID NO: 175 AceIII SEQ ID NO: 157 SEQ ID NO: 176 ApaBI SEQID NO: 157 SEQ ID NO: 177 BbvI SEQ ID NO: 157 SEQ ID NO: 178 BglI SEQ IDNO: 157 SEQ ID NO: 179 BplI/Hin4I SEQ ID NO: 157 SEQ ID NO: 180 BsbI SEQID NO: 157 SEQ ID NO: 181 BtrI SEQ ID NO: 157 SEQ ID NO: 182 HgaI SEQ IDNO: 157 SEQ ID NO: 183 HindIII

In one embodiment, the genetic feature X-2 of the present invention canbe detected, without limitations, by dCAPS method using the followingprimer set and restriction enzyme.

Primer Set:

A primer set comprising a forward primer comprising a sequence (e.g.,SEQ ID NO: 157) of any consecutive 15 bases or more which is positionedupstream of position 108 of SEQ ID NO: 150, and a reverse primercomprising a sequence of any consecutive 15 to 29 bases long from the 3′end of a sequence selected from SEQ ID NOs: 163 and 184 to 191. Thesequences of the primers can be optimized within a range that satisfiesthe conditions described above. For the optimization of primer design,see, for example, Sambrook and Russell (supra). Each of the primers maybe 15 to 50 base long, 18 to 48 base long, 20 to 45 base long, 30 to 65base long, or the like.

Restriction Enzyme:

A restriction enzyme appropriate for each of SEQ ID NOs: 163 and 184 to191 is shown below.

TABLE 3Restriction enzyme appropriate for sequence contained in reverse primerSequence contained in reverse primer Restriction enzymeAAAGTCTCTTGTGTTGAAATTCTGGGGCC (SEQ ID NO: 163) ApaI/BmgI/HgiJIIAAAGTCTCTTGTGTTGAAATTCTGGCGCG (SEQ ID NO: 184) AscI/BglI/BsePIAAAGTCTCTTGTGTTGAAATTCTGGGGCC (SEQ ID NO: 185) AsuI/DraIIAAAGTCTCTTGTGTTGAAATTCTGGCGGC (SEQ ID NO: 186) CviJIAAAGTCTCTTGTGTTGAAATTCTGGGGCG (SEQ ID NO: 187) AcyIAAAGTCTCTTGTGTTGAAATTCTGCCGCT (SEQ ID NO: 188) BsrBIAAAGTCTCTTGTGTTGAAATTCTGGTGCA (SEQ ID NO: 189) MjaIVAAAGTCTCTTGTGTTGAAATTCTGGAGCT (SEQ ID NO: 190) SacIAAAGTCTCTTGTGTTGAAATTCTGGAGAC (SEQ ID NO: 191) EcoPI

In a specific embodiment, the genetic feature X-2 of the presentinvention can be detected by dCAPS method using the following primer setand restriction enzyme.

TABLE 4 Combination of primer set and restriction enzyme Sequence ofSequence of forward primer reverse primer Restriction enzyme SEQ ID NO:157 SEQ ID NO: 163 ApaI/BmgI/HgiJII SEQ ID NO: 157 SEQ ID NO: 184AscI/BglI/BsePI SEQ ID NO: 157 SEQ ID NO: 185 AsuI/DraII SEQ ID NO: 157SEQ ID NO: 186 CviJI SEQ ID NO: 157 SEQ ID NO: 187 AcyI SEQ ID NO: 157SEQ ID NO: 188 BsrBI SEQ ID NO: 157 SEQ ID NO: 189 MjaIV SEQ ID NO: 157SEQ ID NO: 190 SacI SEQ ID NO: 157 SEQ ID NO: 191 EcoPI

In one embodiment, the genetic feature A of the present invention can bedetected, without limitations, by CAPS method using a primer set thatcan amplify a region comprising a sequence shown in any of SEQ ID NOs:19 to 21, and a restriction enzyme that cleaves the polynucleotides ofSEQ ID NOs: 19 to 21 but does not cleave the polynucleotides of SEQ IDNOs: 75 to 77 or a restriction enzyme (e.g., Hpy188I) that does notcleave the polynucleotides of SEQ ID NOs: 19 to 21 but cleaves thepolynucleotides of SEQ ID NOs: 75 to 77. Non-limiting examples of theprimer set include the following.

Forward primer:  (SEQ ID NO: 37) ATGGTTTGGGAATAGCTCTGTTGTT Reverse primer:  (SEQ ID NO: 38) AGAACTTTGTTCTTGAACCTCTTG 

In one embodiment, the genetic feature B of the present invention can bedetected, without limitations, by dCAPS method or the like using thefollowing primer set and a restriction enzyme.

(B-1) a primer set comprising a forward primer comprising the nucleotidesequence shown in SEQ ID NO: 45 and a reverse primer comprising thenucleotide sequence shown in SEQ ID NO: 46;(B-2) a primer set comprising a forward primer comprising the nucleotidesequence shown in SEQ ID NO: 50 and a reverse primer comprising thenucleotide sequence shown in SEQ ID NO: 51;(B-3) a primer set comprising a forward primer comprising the nucleotidesequence shown in SEQ ID NO: 55 and a reverse primer comprising thenucleotide sequence shown in SEQ ID NO: 56; and(B-4) a primer set comprising a forward primer comprising the nucleotidesequence shown in SEQ ID NO: 60 and a reverse primer comprising thenucleotide sequence shown in SEQ ID NO: 61.

However, the primer set is not limited to those having the sequences ofSEQ ID NOs: 45, 46, 50, 51, 55, 56, 60 or 61. For example, the forwardprimer can have in its 3′ end a sequence from the 3′ end of SEQ ID NO:45, 50, 55 or 60 to 15 bases upstream thereof (see the table below), andthe reverse primer can have in its 3′ end a sequence from the 3′ end ofSEQ ID NO: 46, 51, 56 or 61 to 15 bases upstream thereof (see the tablebelow). Such a primer may be 15 to 50 bases long or 20 to 45 bases long.

TABLE 5 Example of primer set Forward primer Reverse primerGenetic feature (sequence from the 3’ end  (sequence from the 3’ end (primer set name) to 15 bases upstream thereof)to 15 bases upstream thereof) B-1 5′-CAAACAACCGGGTAC-3′5′-AGACATTGGCAACTC-3′ (B-1′) (SEQ ID NO: 78) (SEQ ID NO: 79) B-25′-ATTTATTGTATCTAG-3′ 5′-GTACACATGCTACAC-3′ (B-2′) (SEQ ID NO: 80)(SEQ ID NO: 81) B-3 5′-ACGAAACCCGCTTAA-3′ 5′-TAATCCTTGAATTAG-3′ (B-3′)(SEQ ID NO: 82) (SEQ ID NO: 83) B-4 5′-ACACGTATACTAATC-3′5′-CATGGTATGTACAAC-3′ (B-4′) (SEQ ID NO: 84) (SEQ ID NO: 85)

The primer set is not limited to those having the sequences of SEQ IDNOs: 45, 46, 50, 51, 55, 56, 60 or 61. For example, the forward primercan have or comprise a sequence of any 15 or more consecutive bases inSEQ ID NO: 45, 50, 55 or 60, and the reverse primer can have or comprisea sequence of any 15 or more consecutive bases in SEQ ID NO: 46, 51, 56or 61.

(B-1″) A primer set comprising a forward primer having or comprising asequence of any 15 or more consecutive bases in SEQ ID NO: 45 and areverse primer having or comprising a sequence of any 15 or moreconsecutive bases in SEQ ID NO: 46;

(B-2″) a primer set comprising a forward primer having or comprising asequence of any 15 or more consecutive bases in SEQ ID NO: 50 and areverse primer having or comprising a sequence of any 15 or moreconsecutive bases in SEQ ID NO: 51;

(B-3″) a primer set comprising a forward primer having or comprising asequence of any 15 or more consecutive bases in SEQ ID NO: 55 and areverse primer having or comprising a sequence of any 15 or moreconsecutive bases in SEQ ID NO: 56; or

(B-4″) a primer set comprising a forward primer having or comprising asequence of any 15 or more consecutive bases in SEQ ID NO: 60 and areverse primer having or comprising a sequence of any 15 or moreconsecutive bases in SEQ ID NO: 61.

Such a primer may be 15 to 50 bases long, 20 to 45 bases long, or 30 to65 bases long as long as the arbitrary sequence of 15 or moreconsecutive bases is present at the 3′ end.

Examples of the restriction enzymes to be combined with the aboveprimers include the following.

TABLE 6 Restriction enzymes to be combined with primers PrimerRestriction enzyme (B-1), (B-1′), (B-1″) KpnI (B-2), (B-2′), (B-2″) XbaI(B-3), (B-3′), (B-3″) AFIII

In one embodiment, the genetic feature C or D of the present inventioncan be detected by dCAPS method using the following primer set and arestriction enzyme.

Primer set: A primer set comprising a forward primer comprising asequence which is positioned at the 3′ end and selected from SEQ ID NOs:86 to 109, and an optional sequence which is added to the 5′ end of thesequence and is of any consecutive upstream bases following position 28of SEQ ID NO: 6 (e.g., a consecutive sequence of any length), and areverse primer comprising a sequence (e.g., SEQ ID NO: 65 or 110)complementary to a sequence of any consecutive 20 bases or more which ispositioned downstream of position 50 of SEQ ID NO: 6. The sequences ofthe primers can be optimized within a range that satisfies theconditions described above. For the optimization of primer design, see,for example, Sambrook and Russell (supra). Each of the primers may be 15to 50 base long, 18 to 48 base long, 20 to 45 base long, 30 to 65 baselong, or the like.

Restriction Enzyme:

A restriction enzyme appropriate for each of SEQ ID NOs: 86 to 109 isshown below. In the sequences described below, “R” represents A or G,and “Y” represents C or T.

TABLE 7 Restriction enzyme appropriate for sequencecontained in forward primer Sequence contained in  Restrictionforward primer enzyme TTCAGGTAATAAAAGGCCTT  DdeI (SEQ ID NO: 86)TTCAGGTAATAAAAGGCACT  MaeI/SpeI (SEQ ID NO: 87) TTCAGGTAATAAAAGGCTTA AflII/MseI (SEQ ID NO: 88) TTCAGGTAATAAAAGGCTTG  Bce83I (SEQ ID NO: 89)TTCAGGTAATAAAAGGCCTC  BseMII (SEQ ID NO: 90) TTCAGGTAATAAAAGGCACG  BsiI(SEQ ID NO: 91) TTCAGGTAATAAAAGTCATG  BspHI/Hpy178III (SEQ ID NO: 92)TTCAGGTAATAAAAGGCTRT  SfeI (SEQ ID NO: 93) TTCAGGTAATAAAAGGCTTR  SmlI(SEQ ID NO: 94) TTCAGGTAATAAAAGGCAGC  EcoP15I (SEQ ID NO: 95)TTCAGGTAATAAAAGGCYCG  AvaI (SEQ ID NO: 96) TTCAGGTAATAAAAGTGATC  BclI(SEQ ID NO: 97) TTCAGGTAATAAAAGGGAGG  BseRI (SEQ ID NO: 98)TTCAGGTAATAAAAGGCTGC  CviRI/PstI (SEQ ID NO: 99) TTCAGGTAATAAAAGGAACC DrdII (SEQ ID NO: 100) TTCAGGTAATAAAAGGCTGA  Eco57I (SEQ ID NO: 101)TTCAGGTAATAAAAGGCTGG  GsuI (SEQ ID NO: 102) TTCAGGTAATAAAAGGGGTG  HphI(SEQ ID NO: 103) TTCAGGTAATAAAAGGTCTG  Hpy188I (SEQ ID NO: 104)TTCAGGTAATAAAAGGGAAG  MboII (SEQ ID NO: 105) TTCAGGTAATAAAAGGTCGT Pfl1108I (SEQ ID NO: 106) TTCAGGTAATAAAAGTTATA  PsiI (SEQ ID NO: 107)TTCAGGTAATAAAAGGCTCG  TaqI/XhoI (SEQ ID NO: 108) TTCAGGCGATAAAAGGCGTT StySKI (SEQ ID NO: 109) TTCAGGTAATAAAAGGCATT  SpeI (SEQ ID NO: 192)

In a specific embodiment, the genetic feature C or D of the presentinvention can be detected by dCAPS method using the following primer setand restriction enzyme.

TABLE 8 Combination of primer set and restriction enzyme Sequence ofSequence of forward primer reverse primer Restriction enzyme SEQ ID NO:64 SEQ ID NO: 65 SpeI SEQ ID NO: 111 SEQ ID NO: 65 DdeI SEQ ID NO: 112SEQ ID NO: 65 MaeI/SpeI SEQ ID NO: 113 SEQ ID NO: 65 AflII/MseI SEQ IDNO: 114 SEQ ID NO: 65 Bce83I SEQ ID NO: 115 SEQ ID NO: 65 BseMII SEQ IDNO: 116 SEQ ID NO: 65 BsiI SEQ ID NO: 117 SEQ ID NO: 65 BspHI/Hpy178IIISEQ ID NO: 118 SEQ ID NO: 65 SfeI SEQ ID NO: 119 SEQ ID NO: 65 SmlI SEQID NO: 120 SEQ ID NO: 65 EcoP15I SEQ ID NO: 121 SEQ ID NO: 65 AvaI SEQID NO: 122 SEQ ID NO: 65 BclI SEQ ID NO: 123 SEQ ID NO: 65 BseRI SEQ IDNO: 124 SEQ ID NO: 65 CviRI/PstI SEQ ID NO: 125 SEQ ID NO: 65 DrdII SEQID NO: 126 SEQ ID NO: 65 Eco57I SEQ ID NO: 127 SEQ ID NO: 65 GsuI SEQ IDNO: 128 SEQ ID NO: 65 HphI SEQ ID NO: 129 SEQ ID NO: 65 Hpy188I SEQ IDNO: 130 SEQ ID NO: 65 MboII SEQ ID NO: 131 SEQ ID NO: 65 Pfl1108I SEQ IDNO: 132 SEQ ID NO: 65 PsiI SEQ ID NO: 133 SEQ ID NO: 65 TaqI/XhoI SEQ IDNO: 134 SEQ ID NO: 65 StySKI

The screening methods of the present invention may further comprise astep of evaluating an ability to form flower buds in the test steviaplant tissue for which the genetic features of the present inventionhave been detected. The evaluation of the ability to form flower buds isas described in the section relating to the plant of the presentinvention. In this embodiment, the screening method of the presentinvention may be applied to daughter plants obtained by selectingindividuals having a lower ability to form flower buds from among thetest stevia plants in which the genetic feature(s) of the presentinvention is/are detected, and crossing the selected individuals withanother stevia plants. Thus, the screening method of the presentinvention may comprise one or more of the following steps.

(i) Detecting the genetic feature(s) (e.g., the genetic feature X of thepresent invention) of the present invention from the genome of a teststevia plant;

(ii) evaluating the ability to form flower buds in the test stevia planttissue in which the genetic feature(s) has/have been detected;

(iii) selecting an individual having a lower ability to form flower budsfrom among the test stevia plants in which the genetic feature(s) of thepresent invention has/have been detected;

(iv) crossing the selected individual having a lower ability to formflower buds with another stevia plant;

(v) detecting the genetic feature(s) (e.g., the genetic feature X of thepresent invention) of the present invention from the genome of daughterplants obtained by crossing;

(vi) evaluating the ability to form flower buds in the tissue of thedaughter plants in which the genetic feature(s) has/have been detected;and

(vii) selecting individuals having a lower ability to form flower budsfrom among the daughter plants in which the genetic features aredetected.

Individuals having a low ability to form flower buds of choice may be,for example, up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, upto 5%, up to 4%, up to 3%, up to 2%, or up to 1% of the test steviaplants in which the genetic feature(s) of the present invention has/havebeen detected, with respect to the low ability to form flower buds.Other stevia plants to be crossed may or may not contain the geneticfeature(s) of the present invention. In the above embodiment, steps (iv)to (vii) can be repeated a plurality of times. In this way, steviaplants having a lower ability to form flower buds can be screened.

In the screening method of the present invention, the test stevia plantmay be a natural plant or a non-transgenic plant. Non-transgenic plantsare as described in the section relating to the plant of the presentinvention.

In the screening method of the present invention, the test stevia plantmay include a stevia plant subjected to a mutagenesis treatment and aprogeny plant thereof. The mutagenesis treatment is as described in thesection relating to the plant of the present invention, and includestreatment with a mutagen, treatment with radiation or irradiation withlight, and the like.

The present invention also provides the above-mentioned primer set,e.g., the primer set described in Table 2 above, the primer setdescribed in Table 4 above, the primer set comprising the forward primerof SEQ ID NO: 37 and the reverse primer of SEQ ID NO: 38, any one ormore primer set(s) selected from the group consisting of the primer sets(B-1) to (B-4), (B-1) to (B-4′) and (B-1″) to (B-4″) above, and/or theprimer set described in Table 8 above. The present invention furtherprovides a primer set capable of amplifying a region having a nucleotidesequence selected from the group consisting of SEQ ID NOs: 150, 164, 1to 6, 69 to 74 by PCR, for example, a forward primer comprising anucleotide sequence of SEQ ID NO: 151, a primer set with a reverseprimer comprising a nucleotide sequence of SEQ ID NO: 152, a forwardprimer comprising a nucleotide sequence of SEQ ID NO: 7, a primer setwith a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8,a forward primer comprising a nucleotide sequence of SEQ ID NO: 9, aprimer set with a reverse primer comprising a nucleotide sequence of SEQID NO: 10, a forward primer comprising a nucleotide sequence of SEQ IDNO: 11, a primer set with a reverse primer comprising a nucleotidesequence of SEQ ID NO: 12, a forward primer comprising a nucleotidesequence of SEQ ID NO: 13, a primer set with a reverse primer comprisinga nucleotide sequence of SEQ ID NO: 14, a forward primer comprising anucleotide sequence of SEQ ID NO: 15, a primer set with a reverse primercomprising a nucleotide sequence of SEQ ID NO: 16, a forward primercomprising a nucleotide sequence of SEQ ID NO: 17, and a primer set witha reverse primer comprising a nucleotide sequence of SEQ ID NO: 18.

In addition, the present invention provides a probe capable of detectingthe presence and/or absence of the genetic features of the presentinvention, which may be referred to as the “probe of the presentinvention” hereinafter. The probe of the present invention may have astructure suitable for various detection methods for the presence and/orabsence of the genetic feature(s) of the present invention. For example,the probe of the present invention may comprise a nucleotide sequencecomplementary to a portion of a genome comprising a variation site ofthe present invention. Non-limiting examples of such probes includethose comprising a nucleotide sequence selected from SEQ ID NOs: 153 to156, 168 to 171, 19 to 36, 75 to 77, 135 to 149. Of these sequences, SEQID NOs: 153 to 156, 19 to 36 are specific for alleles comprising thevariation of the present invention, and SEQ ID NOs: 168 to 171, 75 to77, 135 to 149 are specific for alleles not containing the variation ofthe present invention. The presence of the genetic feature(s) of thepresent invention may be detected by detection of an allele comprisingthe variation(s) of the present invention and/or by non-detection of anallele not comprising the variation(s) of the present invention, and theabsence of the genetic feature(s) of the invention by non-detection ofan allele comprising the variation(s) of the present invention and/or bydetection of an allele not comprising the variation(s) of the presentinvention. The probes of the present invention preferably have a label.Non-limiting examples of such labels include fluorescent labels,luminescent labels, radioactive labels, dyes, enzymes, quenchers,binding moieties with detectable labels, and the like. In a specificembodiment, the probe of the present invention has a nucleotide sequencecomplementary to the nucleotide sequence selected from SEQ ID NOs: 153to 156, 168 to 171, 19 to 36, 75 to 77, 135 to 149 and a label.

The present invention further provides a kit, for example, a kit forscreening, comprising a primer set comprising a forward primercomprising a sequence (e.g., SEQ ID NO: 157) of any consecutive 15 basesor more which is positioned upstream of position 90 of SEQ ID NO: 150,and a reverse primer comprising a sequence of any consecutive 15 to 25bases long from the 3′ end of a sequence selected from SEQ ID NOs: 158and 172 to 183, for example, a primer set described in Table 2, and arestriction enzyme appropriate therefor.

The present invention further provides a kit, for example, a kit forscreening, comprising a primer set comprising a forward primercomprising a sequence (e.g., SEQ ID NO: 157) of any consecutive 15 basesor more which is positioned upstream of position 108 of SEQ ID NO: 150,and a reverse primer comprising a sequence of any consecutive 15 to 25bases long from the 3′ end of a sequence selected from SEQ ID NOs: 163and 184 to 191, for example, a primer set described in Table 4, and arestriction enzyme appropriate therefor.

The present invention further provides a kit, for example, a kit forscreening, comprising a primer set that can amplify a region comprisinga sequence shown in any of SEQ ID NOs: 19 to 21, for example, a primerset comprising the combination of a forward primer comprising thenucleotide sequence of SEQ ID NO: 7 and a reverse primer comprising thenucleotide sequence of SEQ ID NO: 8, and a restriction enzyme thatcleaves the polynucleotides of SEQ ID NOs: 19 to 21 but does not cleavethe polynucleotides of SEQ ID NOs: 75 to 77 or a restriction enzyme(e.g., Hpy188I) that does not cleave the polynucleotides of SEQ ID NOs:19 to 21 but cleaves the polynucleotides of SEQ ID NOs: 75 to 77.

The present invention further provides a kit, for example, a kit forscreening comprising any one or more primer set(s) selected from thegroup consisting of the primer sets (B-1) to (B-4), (B-1′) to (B-4′) and(B-1″) to (B-4″), and optionally a restriction enzyme.

In the kit, the restriction enzyme contained in the kit is KpnI in thecase of using any one or more primer set(s) selected from the groupconsisting of the primer sets (B-1), (B-1′) and (B-1″).

In the kit, the restriction enzyme contained in the kit is XbaI in thecase of using any one or more primer set(s) selected from the groupconsisting of the primer sets (B-2), (B-2′) and (B-2″).

In the kit, the restriction enzyme contained in the kit is AflII in thecase of using any one or more primer set(s) selected from the groupconsisting of the primer sets (B-3), (B-3′) and (B-3″).

The present invention further provides a kit, for example, a kit forscreening, comprising a primer set comprising a forward primercomprising a sequence which is positioned at the 3′ end and selectedfrom SEQ ID NOs: 86 to 109 and 192, and an optional sequence which isadded to the 5′ end of the sequence and is of any consecutive upstreambases following position 28 of SEQ ID NO: 6 (e.g., a consecutivesequence of any length), and a reverse primer comprising a sequence(e.g., SEQ ID NO: 65 or 110) complementary to a sequence of anyconsecutive 20 bases or more which is positioned downstream of position50 of SEQ ID NO: 6, for example, a primer set described in Table 8, anda restriction enzyme appropriate therefor.

In Another Embodiment of the Kit:

in case the primer set comprises a forward primer having or comprising asequence of any consecutive 15 bases or more in SEQ ID NO: 45, therestriction enzyme comprises KpnI;

in case the primer set comprises a forward primer having or comprising asequence of any contiguous 15 bases or more in SEQ ID NO: 50, therestriction enzyme comprises XbaI; and

in case the primer set comprises a forward primer having or comprising asequence of any consecutive 15 bases or more in SEQ ID NO: 55, therestriction enzyme comprises AflII.

The present invention also provides a screening kit comprising a primerset capable of amplifying by PCR a region having a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 153 to 156, 168 to171, 1 to 6, 19 to 36, 69 to 77, 135 to 149, and a probe of the presentinvention.

These primer sets, probes and kits can be used to detect the geneticfeature(s) of the present invention, used in the screening methods ofthe present invention, and the like. These primer sets and kits may alsocomprise an instruction including an explanation on the detection ofgenetic feature(s) of the present invention and on the screening methodof the present invention, e.g., a written instruction, and media, e.g.,a flexible disk, a CD, a DVD, a Blu-ray disk, a memory card, a USBmemory, etc., having recorded thereon information regarding the methodof use.

4. Method of Producing Extract Derived from Plant and Product Comprisingthe Extract

In a further aspect, the present invention provides a method ofproducing a stevia extract, comprising a step of obtaining an extractfrom the plant of the present invention, or a seed or a leaf (e.g.,dried leaf or fresh leaf) of the plant (hereinafter, may be referred toas the “extract production method of the present invention”). Thepresent invention further provides a method of producing a steviolglycoside purified product, comprising a step of purifying a steviolglycoside from an extract obtained by the extract production method ofthe present invention (hereinafter, may be referred to as the “steviolglycoside purified product production method of the present invention”).

Specifically, the present invention provides a method of producing asteviol glycoside purified product, comprising a step of obtaining anextract containing a steviol glycoside from the stevia plant of thepresent invention, the stevia plant screened for by the screening methodof the present invention, or the stevia plant produced by the method ofthe present invention, and a step of purifying a steviol glycoside fromthe obtained extract.

The extract containing a steviol glycoside can be obtained by reacting afresh leaf or a dried leaf of the plant of the present invention with asuitable solvent (an aqueous solvent such as water or an organic solventsuch as an alcohol, ether or acetone). For the extraction conditions,etc., see a method described in Ohta et al. (supra) or WO2010/038911, ora method described in Examples mentioned later.

Individual steviol glycoside can be purified from the extract containinga steviol glycoside by use of a method known in the art such as agradient of ethyl acetate or any of other organic solvents:water, highperformance liquid chromatography (HPLC), gas chromatography,time-of-flight mass spectrometry (TOF-MS), or ultra (high) performanceliquid chromatography (UPLC).

Examples of the steviol glycoside include RebA, RebB, RebC, RebD, RebE,RebF, Rebl, RebJ, RebK, RebM, RebN, RebO, RebQ, RebR, dulcoside A,rubusoside, steviolmonoside, steviolbioside, and stevioside. In oneembodiment, the steviol glycoside includes RebA, RebB, RebC, RebD, RebE,RebF, RebM, RebN, RebO, stevioside, steviolbioside, rubusoside,dulcoside A or a combination thereof. In a preferable embodiment, thesteviol glycoside includes RebD, RebM or a combination thereof.

One form of the extract obtained by the extract production method of thepresent invention (hereinafter, may be referred to as the “extract ofthe present invention”) comprises RebD or RebM, or both at highercontent as compared with the wild type stevia species.

The extract of the present invention may comprise RebD or RebM, or bothat a content higher by 300% or more, 400% or more, 500% or more, 600% ormore, 700% or more, 800% or more, 900% or more, 1100% or more, 1200% ormore, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700%or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more,2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% ormore, 2700% or more, 2800% or more, 2900% or more, 3000% or more, 3100%or more, 3200% or more, 3300% or more, 3400% or more, 3500% or more,3600% or more, 3700% or more, 3800% or more, 3900% or more, 4000% ormore, 4100% or more, 4200% or more, 4300% or more, 4400% or more, 4500%or more, 4600% or more, 4700% or more, 4800% or more, 4900% or more,5000% or more as compared with an extract obtained from the wild typestevia species. The extract of the present invention and the extractobtained from the wild type stevia species may be those obtained by thesame process.

The extract of the present invention thus obtained and/or the steviolglycoside purified product (e.g., RebD and/or RebM) obtained by themethod of producing a steviol glycoside purified product according tothe present invention can be mixed with other component(s) to produce amedicament, flavor or food or beverage containing a steviol glycoside.Accordingly, in an alternative aspect, the present invention provides amethod of producing a medicament, a flavor or a food or beverage,comprising a step of mixing the extract of the present invention and/orthe steviol glycoside purified product obtained by the method ofproducing a steviol glycoside purified product according to the presentinvention with other component(s). The present invention furtherprovides a medicament, flavor or food or beverage containing a steviolglycoside, obtained by the production method. In this context, the foodor beverage means a drink and a food. Thus, in a certain embodiment, thepresent invention provides a medicament, flavor, drink or food and alsoprovides a method of producing the medicament, the flavor, the drink orthe food.

5. Nucleotide Sequence Related to Plant of Present Invention

In another aspect, the present invention provides a nucleotide sequencerelated to the stevia plant of the present invention.

The nucleotide sequence related to a stevia plant having the geneticfeature X-1 comprises or consists of a nucleotide sequence selected fromSEQ ID NOs: 153 and 154. The nucleotide sequence related to a steviaplant having the genetic feature X-2 comprises or consists of anucleotide sequence selected from SEQ ID NOs: 155 and 156. Thenucleotide sequence related to a stevia plant having the genetic featureA comprises or consists of a nucleotide sequence selected from SEQ IDNOs: 19 to 21 and 69. The nucleotide sequence related to a stevia planthaving the genetic feature B-1 comprises or consists of a nucleotidesequence selected from SEQ ID NOs: 22 to 24 and 70. The nucleotidesequence related to a stevia plant having the genetic feature B-2comprises or consists of a nucleotide sequence selected from SEQ ID NOs:25 to 27 and 71. The nucleotide sequence related to a stevia planthaving the genetic feature B-3 comprises or consists of a nucleotidesequence selected from SEQ ID NOs: 28 to 30 and 72. The nucleotidesequence related to a stevia plant having the genetic feature B-4comprises or consists of a nucleotide sequence selected from SEQ ID NOs:31 to 33, 62 and 73. The nucleotide sequence related to a stevia planthaving the genetic feature C or D comprises or consists of a nucleotidesequence selected from SEQ ID NOs: 34 to 36 and 74.

EXAMPLES

Hereinafter, the present invention will be described with reference toExperimental Examples, Examples, etc. However, the present invention isnot limited by these specific embodiments.

[Example 1] Verification of Relationship Between RebM Content andGenetic Feature B—(1)

Individuals derived from commercially available stevia seeds weresubjected to selection based on the development and growth condition,the foliar morphology, the contents of total steviol glycoside (TSG),RebA, RebD, and RebM, etc. to obtain two segregating populations, i.e.,individual groups I and II. 62 individuals from the individual group Iand 109 individuals from the individual group II were used inverification. Each individual group was divided on the basis of RebMcontent into 3 groups: 0.2% or more, 0.1% or more to less than 0.2%, and0% or more to less than 0.1%, and examined for the presence or absenceof the genetic feature B-1. Specifically, PCR was performed using theprimers given below. A restriction enzyme (KpnI) was added to the PCRproduct, and enzymatic reaction was performed at 37° C. for treatmentwith the restriction enzyme. After the restriction enzyme treatment,electrophoresis was performed using a microchip type electrophoresisapparatus LabChip GX Touch HT. The marker was identified on the basis ofa band pattern after the electrophoresis.

The primer sequences are as follows.

Fw primer:  (SEQ ID NO: 45)5′-TAATCATCCAAACCCTAATCTCGCCAAACAACCGGGTAC-3′ Rv primer:(SEQ ID NO: 46)  5′-GAGGAAGACATTGGCAACTC-3′

When a restriction enzyme-treated product of approximately 260 bp (e.g.,SEQ ID NO: 49) was not formed by the KpnI restriction enzyme treatmentof the obtained PCR product (approximately 297 bp long), the testsubject was regarded as being positive for B-1. As a result, the groupcontaining 0.2% or more or RebM was preferentially detected with thegenetic feature of the present invention. These results demonstratedthat the frequency of appearance of positive individuals wasstatistically significantly different among groups (goodness of fit testby the chi square test; the null hypothesis stated that the frequencydistribution was even without the association of the marker test resultswith the phenotype; for the test results, see the tables below).

TABLE 9 Results of test on individual group I (62 individuals) Testresults RebM content (%) Positive Negative Total 0.2 or more 29 8 37 0.1or more and less than 0.2 0 20 20 0 or more and less than 0.1 0 5 5Total 29 33 62 Chi square test result (df = 2) 36.81**

TABLE 10 Results of test on individual group II (109 individuals) Testresults RebM content (%) Positive Negative Total 0.2 or more 18 1 19 0.1or more and less than 0.2 35 3 38 0 or more and less than 0.1 5 47 52Total 58 51 109 Chi square test result (df = 2) 75.94**

[Example 2] Verification of Relationship Between RebM Content andGenetic Feature B (2)

As a result of selecting a plant with high RebM content using thegenetic feature B, individuals having a RebM ratio of 2% or more werealso able to be selected in segregating populations other than thepopulations for verification, as shown in the table below, confirmingthat these genetic markers are capable of serving as practical selectionmarkers. The results of selecting a plant with high RebM content areshown in the table below. In the table, the circle mark represents thatthe genetic feature B test results were positive.

The genetic feature B-1 was detected in the same way as in Example 1,and the genetic features B-2 to B-4 were detected as follows.

For the detection of the genetic feature B-2, PCR was performed usingthe primers given below. A restriction enzyme (XbaI) was added to thePCR product, and enzymatic reaction was performed at 37° C. fortreatment with the restriction enzyme. After the restriction enzymetreatment, electrophoresis was performed using a microchip typeelectrophoresis apparatus LabChip GX Touch HT. The marker was identifiedon the basis of a band pattern after the electrophoresis.

The primer sequences are as follows.

Fw primer: (SEQ ID NO: 50) 5′-AAGGTTCTTTATTTTTAAACTTATGTTAATTTATTGTATCTAG-3′ Rv primer:(SEQ ID NO: 51)  5′-CCTTATGTACACATGCTACAC-3′

When a restriction enzyme-treated product of approximately 344 bp (e.g.,SEQ ID NO: 54) was not formed by the XbaI restriction enzyme treatmentof the obtained PCR product (approximately 383 bp long), the testsubject was regarded as being positive for the genetic feature B-2.

For the detection of the genetic feature B-3, PCR was performed usingthe primers given below. A restriction enzyme (AflII) was added to thePCR product, and enzymatic reaction was performed at 37° C. fortreatment with the restriction enzyme. After the restriction enzymetreatment, electrophoresis was performed using a microchip typeelectrophoresis apparatus LabChip GX Touch HT. The marker was identifiedon the basis of a band pattern after the electrophoresis.

The primer sequences are as follows.

Fw primer:  (SEQ ID NO: 55) 5′-CGATGGTTTTTGCTACATGAAAACCCTAGAAGACGAAACCCGC TTAA-3′ Rv primer:(SEQ ID NO: 56)  5′-ACCAGCAATAATCCTTGAATTAG-3′

When a restriction enzyme-treated product of approximately 347 bp (e.g.,SEQ ID NO: 59) was not formed by the AflII restriction enzyme treatmentof the obtained PCR product (approximately 390 bp long), the testsubject was regarded as being positive for the genetic feature B-3.

For the detection of the genetic feature B-4, PCR was performed usingthe primers given below. The PCR product was electrophoresed using amicrochip type electrophoresis apparatus LabChip GX Touch HT. The markerwas identified on the basis of a band pattern after the electrophoresis.

The primer sequences are as follows.

Fw primer: (SEQ ID NO: 60)  5′-CGCAAACACGTATACTAATC-3′ Rv primer:(SEQ ID NO: 61)  5′-TTTAGCATGGTATGTACAAC-3′

When only a PCR product of approximately 140 bp (e.g., SEQ ID NO: 62)was formed, the test subject was regarded as being positive for B-4.

TABLE 11 RebA RebD RebM RebA + RebM T

Content Content Content Content (%) (%) RA/TSG (%) RD/TSG (%) RM/TSG (%)RAM/TSG

1736 18.45

71.73% 1.77 9.58% 1.46 7.92% 14.69 79.65%

7518 15.10 7.20 47.64% 3.57 23.64% 1.29 8.56% 8.49 56.20%

8.96 5.59 62.37% 1.42 15.85% 1.19 13.31% 6.78 75.68%

9.12 6.07 66.53% 1.09 11.91% 1.11 12.14% 7.18 78.67%

1749

4.16 56.20% 1.42 19.23% 1.07 14.49% 5.23 70.69%

21817

4.46 61.32% 1.13 16.50% 1.03 14.11% 5.49 75.43%

21745 9.53 6.91 66.22% 1.32 13.83% 0.97 10.13% 7.28 76.36%

21706 8.77 5.26 59.97% 1.61 18.30% 0.96 11.00% 6.22 70.97%

21714 9.26 5.47 59.06% 1.97 21.30% 0.94 10.10% 6.41 69.16%

21707 9.76 6.23 63.82% 1.57 16.09% 0.92 9.45% 7.15 79.27%

21750 8.11 5.21 64.25% 1.15 14.20% 0.90 11.13% 6.11 75.38%

21751 8.25 5.32 64.24% 1.27 15.32% 0.90 10.89% 8.22 75.19%

7535 11.61 6.54 56.35% 2.25 19.42% 0.90 7.75% 7.44 64.10%

21818 7.09 4.32 61.28% 1.14 16.16% 0.89 12.66% 5.21 73.94%

21246 8.66 5.19 59.91% 1.68 19.34% 0.59 10.27% 6.08 70.18%

9.83 5.46 55.57% 2.16 21.96% 0.87 8.88% 6.33 64.44%

21796 8.27 5.30 64.08% 1.33 16.11% 0.85 10.30% 0.15 74.38%

7567 7.86 3.92 49.34% 2.15 27.30% 0.85 10.79% 4.77 80.69%

21811 9.22 6.08 65.43% 1.42 15.41% 0.85 9.19% 5.88 74.62%

21742 9.37 6.39 68.22% 1.35 14.35% 0.74 7.93% 7.13 76.15%

1753 6.70 3.60 53.75% 1.68 25.08% 0.68 9.78% 4.26 63.54%

7565 8.91 3.21 36.02% 2.77 31.11% 0.59 5.96% 3.74 41.98%

21361 18.28 11.97 65.48% 2.52 13.81% 0.46 2.51% 12.42 67.99%

21255 15.09 9.78 64.30% 1.84 12.20% 0.35 2.34% 10.13 67.15%

21356 13.09 7.83 59.84% 2.16 16.49% 0.31 2.36% 8.14 62.20%

21357 13.66 9.24 67.64% 1.66 12.18% 0.30 2.21% 9.54 69.85%

21375 14.04 8.61 61.33% 2.32 16.50% 0.30 2.12% 8.91 63.45%

21369 12.68 7.89 62.34% 1.58 12.49% 0.29 2.25% 8.18 64.60%

21360 12.68 7.86 61.95% 1.82 14.31% 0.29 2.25% 8.15 64.22%

21358 10.48 6.28 59.95% 1.83 16.05% 0.27 2.57% 6.55 82.52%

1785 5.50 1.94 35.25% 1.61 29.18% 0.24 4.39% 2.18 39.64%

14252 6.93 2.05 29.74% 1.66 29.99% 0.21 3.02% 2.27 32.77%

17197 7.97 2.65 33.26% 1.72 21.59% 0.19 2.37% 2.94 36.63%

21314 11.68 7.57 64.83% 0.10 0.83% 0.04 0.33% 7.61 65.15%

1552 13.20 8.94 67.72% 0.09 0.71% 0.04 0.27% 8.97 67.99%

21297 11.57 6.90 59.66% 0.11 0.91% 0.03 0.26% 8.93 59.92%

1357 10.43 6.04 57.89% 0.11 1.03% 0.03 0.25% 6.06 58.14%

7546 11.30 3.46 30.82% 0.08 0.89% 0.01 0.06% 3.49 30.88% RebD + RebMContent (%) RDM/TSG RebM/RebA RebM/RebD P01 P02 P03 P04 P05

1736 9.23 17.51% 0.11 0.83 ◯ ◯ ◯ ◯ ◯

7518 4.86 32.19% 0.18 0.36 ◯ ◯ ◯ ◯ ◯

2.61 29.16% 0.21 0.84 ◯ ◯ ◯ ◯ ◯

2.19 24.05% 0.16 1.02 ◯ ◯ ◯ ◯ ◯

1749 2.50 33.73% 0.26 0.75 ◯ ◯ ◯ ◯ ◯

21817 2.15 29.61% 0.23 0.91 ◯ ◯ ◯ ◯ ◯

21745 2.25 23.96% 0.15 0.73 ◯ ◯ ◯ ◯ ◯

21706 2.57 29.30% 0.18 0.60 ◯

21714 2.91 31.40% 0.17 0.47 ◯ ◯ ◯ ◯ ◯

21707 2.49 28.54% 0.15 0.59 ◯ ◯ ◯ ◯ ◯

21750 2.05 25.33% 0.17 0.78 ◯ ◯ ◯ ◯ ◯

21751 2.17 26.21% 0.17 0.71 ◯ ◯ ◯ ◯ ◯

7535 3.15 27.17% 0.14 0.40 ◯ ◯ ◯ ◯ ◯

21818 2.03 28.82% 0.21 0.78 ◯ ◯ ◯ ◯ ◯

21246 2.57 29.61% 0.17 0.53 ◯ ◯ ◯ ◯ ◯

751 3.03 30.84% 0.16 0.40 ◯ ◯ ◯ ◯

21796 2.18 28.41% 0.16 0.61 ◯ ◯ ◯ ◯ ◯

7567 2.99 38.09% 0.22 0.40 ◯ ◯ ◯ ◯ ◯

21811 2.27 24.61% 0.14 0.60 ◯ ◯ ◯ ◯ ◯

21742 2.09 22.30% 0.12 0.55 ◯ ◯ ◯ ◯ ◯

1753 2.33 34.86% 0.14 0.39 ◯ ◯ ◯ ◯ ◯

7565 3.30 37.07% 0.17 0.19 ◯ ◯ ◯ ◯

21361 2.98 16.33% 0.04 0.18 ◯ ◯ ◯ ◯ ◯

21255 2.20 14.55% 0.04 0.19 ◯ ◯ ◯ ◯ ◯

21356 2.47 18.85% 0.04 0.14 ◯ ◯ ◯ ◯ ◯

21357 1.97 14.39% 0.03 0.18 ◯ ◯ ◯ ◯ ◯

21375 2.63 18.62% 0.03 0.18 ◯ ◯ ◯ ◯ ◯

21369 1.87 14.74% 0.04 0.18 ◯ ◯ ◯ ◯ ◯

21360 2.10 16.56% 0.04 0.16 ◯ ◯ ◯ ◯ ◯

21358 1.95 18.64% 0.04 0.16 ◯ ◯ ◯ ◯ ◯

1785 1.85 33.57% 0.12 0.15 ◯ ◯ ◯ ◯ ◯

14252 1.87 27.01% 0.10 0.13 ◯ ◯ ◯ ◯

17197 1.91 23.97% 0.07 0.11 ◯ ◯ ◯ ◯ ◯

21314 0.14 1.17% 0.01 0.38

1552 0.13 0.98% 0.00 0.38

21297 0.14 1.17% 0.00 0.28

1357 0.13 1.28% 0.00 0.24

7546 0.09 0.75% 0.00 0.09

indicates data missing or illegible when filed

[Example 3] Verification of Relationship Between Stevia Plant with HighTSG Content and Genetic Feature C

(1) Isolation of Individual with High Sweet Content (M0 Generation)

Approximately 2000 (based on weight) wild type stevia seeds (commercialvariety; introduced in August 2014) were divided into 3 groups, each ofwhich was genetically modified by a treatment with 0.1%, 0.2% or 0.3%ethylmethanesulfonic acid (EMS).

The seeds thus treated with EMS and untreated seeds were seeded in agreenhouse within the Suntory research center to obtain EMS-treatedgeneration (M0 generation) seedlings. No difference in the rate ofgermination was seen among the treatment concentrations.

An appropriate amount of fresh leaves was sampled from the EMS-treatedgeneration (M0 generation) and untreated individuals, and theconcentration of a sweet component was quantitatively determined byLC/MS-MS (Shimadzu LCMS8050). Specifically, 0.25 g of fresh leaves wasdried by freeze drying, and 0.05 g of homogenized dry matter thereof wasadded into pure water. Extraction by ultrasonic treatment for 20minutes, and centrifugation and filtration were performed to obtain 0.33ml of a liquid extract. The concentrations of RebA, RebB, RebC, RebD,RebF, RebM, RebN and RebO were quantitatively determined by LC/MS-MSanalysis on this liquid extract in a LCMS8050 ion mode (ShimadzuLCMS8050), and the total sum thereof was regarded as the concentrationof the sweet component. Individuals having a sweet componentconcentration of approximately 20% were used as parent individuals 1(P1). Also, individuals which were derived from other populations ofstevia plants and had a sweet component concentration of 5% in a driedleaf were selected as parent individuals 2 (P2).

(2) Isolation of High Sweet Content Individual (M1 Generation) and GeneAnalysis

The first treated generation (M1 generation) seeds were produced by thecrossing of the parent individuals 1 (P1) with the parent individuals 2(P2), and seeded in a greenhouse within the Suntory research center toobtain M1 generation seedlings (segregating population of 1603individuals). An appropriate amount of fresh leaves was sampled from theM1 generation individuals, and the concentration of a sweet componentwas quantitatively determined by LC/MS-MS (Shimadzu LCMS8050) in thesame way as above. The results are shown in FIG. 1.

Genomic DNA was extracted from fresh leaves of 30 individuals withhighest content of the sweet component (high sweet content individuals)and 30 individuals with lowest content of the sweet component (low sweetcontent individuals), and examined for a variation present only in anyone of the individual groups. Among variations detected by the genomicanalysis, 306 variations which had a sufficient amount of genomicinformation (sequence coverage: x5 or more), had no continuation ofvariations, and were free from a sequence insertion or deletion wereeach studied for an individual in which the variation was present. As aresult, a variation from C to A (C49A) at position 49 of SEQ ID NO: 1was found to be present in the high sweet content individuals, but beabsent in the low sweet content individuals.

(3) Verification of Relationship Between Variation C49A and SweetContent

Stevia plants heterozygously having the variation C49A were crossed withstevia plants lacking the variation C49A to obtain two segregatingpopulations (segregating population A (443 individuals) and segregatingpopulation B (446 individuals)). The presence or absence of thevariation C49A in each individual of both the segregating populations,and a sweet content were examined. The dCAPS method was used in theexamination of the presence or absence of the variation C49A. GenomicDNA was extracted from each individual, and PCR was performed using theprimers given below. A restriction enzyme (SpeI) was added to the PCRproduct, and enzymatic reaction was performed at 37° C. After therestriction enzyme treatment, electrophoresis was performed using amicrochip type electrophoresis apparatus LabChip GX Touch HT(PerkinElmer). The marker was identified on the basis of a band patternafter the electrophoresis.

Forward primer: (SEQ ID NO: 112) 5′-TTATTTAATGATCCAATGGAGGGGGTGATTCAGGTAATAAAAGG CACT-3′ Reverse primer:(SEQ ID NO: 65) 5′-TGAGGGTTCTCAATTGATTTCCGATTGG-3′

When a restriction enzyme-treated product of approximately 321 bp (e.g.,SEQ ID NO: 68) was formed by the SpeI restriction enzyme treatment ofthe obtained PCR product of approximately 367 bp (e.g., SEQ ID NO: 66 or67), the test subject was regarded as being positive for the variationC49A.

The quantitative determination of the sweet content was performed in thesame way as in (1). The distribution of sweet contents of variation C49Apositive individuals and negative individuals of each segregatingpopulation is shown in FIGS. 2 and 3. As is evident from these results,the sweet contents of the variation C49A positive individuals werehigher than the average sweet content of each whole segregatingpopulation.

The average and median sweet contents of the variation C49A positiveindividuals and negative individuals in each segregating population aresummarized below.

TABLE 12 Average and median sweet component concentrations (%) in driedleaves of each segregating population Average (%) Median (%) Segregatingpopulation A Total 12.3 10.4 C49A⁺ 17.4 17.3 C49A⁻ 7.7 7.5 Segregatingpopulation B Total 5.5 3.7 C49A⁺ 9.8 10.0 C49A⁻ 3.3 3.2

[Example 4] Verification of Relationship Between Stevia Plant with HighRebD Content and Genetic Features A to C

1. Production of Test Lines

Male stocks (P1) having a high TSG-content genetic feature were crossedwith female stocks (P2) having a high RebM-content genetic feature toproduce the first filial generation (S1 generation) seeds. The seedswere seeded in a greenhouse within the Suntory research center to obtainS1 generation seedlings.

The high RebM-content genetic feature has at least one of the followingfeatures.

B-1: Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T.

B-2: Homozygous for the allele wherein the base at the positioncorresponding to position 44 of SEQ ID NO: 3 is T.

B-3: Homozygous for the allele wherein the base at the positioncorresponding to position 41 of SEQ ID NO: 4 is C.

B-4: Homozygous for the allele wherein the portion corresponding topositions 55-72 of SEQ ID NO: 5 is deleted.

As shown in Examples 1 and 2, these genetic features are related to thehigh RebM-content characteristics.

The high TSG-content genetic feature has the following feature.

C: Heterozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A.

As shown in Example 3, the genetic feature is related to the highTSG-content characteristics.

Both P1 and P2 are progeny of individuals genetically modified byethylmethanesulfonic acid (EMS) treatment.

An appropriate amount of fresh leaves was sampled from the P1, P2 and S1generation individuals, 0.25 g of fresh leaves was dried by freezedrying, and 0.05 g of homogenized dry matter thereof was added into purewater. Extraction by ultrasonic treatment for 20 minutes, andcentrifugation and filtration were performed to obtain 0.33 ml of aliquid extract. The concentrations (% by mass with respect to a driedleaf) of RebA, RebB, RebC, RebD, RebF, RebM, RebN and RebO werequantitatively determined by LC/MS-MS analysis on this liquid extract ina LCMS8050 ion mode (Shimadzu LCMS8050), and the total sum thereof wasregarded as the concentration of total steviol glycoside (TSG). Theresults are shown in the table below.

TABLE 13 Glycoside concentration in crossing parents and S1 generationindividuals RebD + ID RebA RebD RebD/TSG RebM RebM/TSG RebD + RebMRebM/TSG TSG number (% by mass) (% by mass) (%) (% by mass) (%) (% bymass) (%) (% by mass) P1 16.9 1.0 2.8 0.1 0.3 1.1 3.1 36 P2 2.8 0.5 12.20.4 9.8 0.9 22.0 4.1 S1-1 3.5 3.9 33.9 0.6 5.2 4.5 39.1 11.5 S1-2 4.03.6 31.9 0.7 6.2 4.3 38.1 11.3 S1-3 6.1 3.4 27.9 0.8 6.6 4.2 34.4 12.2S1-4 6.8 2.5 20.5 0.6 4.9 3.1 25.4 12.2 S1-5 6.8 2.4 20.5 0.7 6.0 3.126.5 11.7 S1-6 7.2 1.5 13.6 1.0 9.1 2.5 22.7 11 S1-7 9.2 0.8 6.7 0.5 4.21.3 10.9 11.9 S1-8 6.2 0.8 5.6 0.1 0.7 0.9 6.3 14.4 S1-9 6.5 0.8 5.1 0.10.6 0.9 5.7 15.7

As shown in the results, high RebD-content individuals having a RebDcontent exceeding 3.3% by mass based on a dried leaf were obtained (S1-1to S1-3) by the crossing of P1 with P2.

[Example 5] Detection of Genetic Feature Unique to Stevia Plant withHigh RebD Content

Genomic DNA was extracted from the fresh leaves of each individualtested in Example 4, and examined for the condition retaining thegenetic features B-1 and C.

For the detection of the genetic feature B-1, PCR was performed usingthe primers given below. A restriction enzyme (KpnI) was added to thePCR product, and enzymatic reaction was performed at 37° C. fortreatment with the restriction enzyme. After the restriction enzymetreatment, electrophoresis was performed using a microchip typeelectrophoresis apparatus LabChip GX Touch HT. The marker was identifiedon the basis of a band pattern after the electrophoresis.

The primer sequences are as follows.

Fw primer: (SEQ ID NO: 45) 5′-TAATCATCCAAACCCTAATCTCGCCAAACAACCGGGTAC-3′ Rv primer:(SEQ ID NO: 46)  5′-GAGGAAGACATTGGCAACTC-3′

When a restriction enzyme-treated product of approximately 260 bp (e.g.,SEQ ID NO: 49) was not formed by the KpnI restriction enzyme treatmentof the obtained PCR product (approximately 297 bp long), the testsubject was regarded as being positive for the genetic feature B-1.

For the detection of the genetic feature C, PCR was performed using theprimers given below. A restriction enzyme (SpeI) was added to the PCRproduct, and enzymatic reaction was performed at 37° C. for treatmentwith the restriction enzyme. After the restriction enzyme treatment,electrophoresis was performed using a microchip type electrophoresisapparatus LabChip GX Touch HT (PerkinElmer). The marker was identifiedon the basis of a band pattern after the electrophoresis.

Forward primer: (SEQ ID NO: 112)5′-TTATTTAATGATCCAATGGAGGGGGTGATTCAGGTAATAAAAG GCACT-3′ Reverse primer: (SEQ ID NO: 65) 5′-TGAGGGTTCTCAATTGATTTCCGATTGG-3′

When a restriction enzyme-treated product of approximately 321 bp (e.g.,SEQ ID NO: 68) was formed by the SpeI restriction enzyme treatment ofthe obtained PCR product of approximately 367 bp (e.g., SEQ ID NO: 66 or67), the test subject was regarded as being positive for the geneticfeature C.

The results are shown in Table 14 below. In the table, the circle markrepresents that the corresponding variation was detected, and the x markrepresents that the corresponding variation was not detected.

TABLE 14 Genetic feature ID number B-1 C P1 x ∘ P2 ∘ x S1-1 ∘ ∘ S1-2 ∘ ∘S1-3 ∘ ∘ S1-4 ∘ ∘ S1-5 ∘ ∘ S1-6 ∘ ∘ S1-7 ∘ ∘ S1-8 x ∘ S1-9 x ∘

As shown in the results, the individuals retaining the genetic featureB-1 tended to have a higher RebM content than that of the individualsnot retaining the genetic feature B-1, and the individuals retaining thegenetic feature C tended to have higher TSG content than that of theindividuals not retaining the genetic feature C. This supports theresults shown in the earlier applications by the present applicant.

In order to find a marker for identifying individuals with high RebDcontent, genomic DNA was extracted from the fresh leaves of eachindividual and sequenced with NGS (HiSeq 2500, Illumina, Inc.). As aresult, the following genetic feature was found only in the individualswith high RebD content.

A: Homozygous for the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A.

As shown in the results, all the individuals with high RebD content(S1-1 to S1-3) retained the genetic features A, B-1 and C and tended tohave a higher RebD content than that of the individuals not retainingthe genetic feature A.

[Example 6] Verification of Relationship Between Ability to Form FlowerBuds and Genetic Feature X

After cutting of two wild type stevia plant lines (W101 and W102) andone variant stevia plant line (M101) which was progeny of individualsgenetically modified by a mutagenesis treatment withethylmethanesulfonic acid (EMS), plants each having 8 true leaves wereplaced under each condition shown in the table below. Three weeks later,the number of flower buds was counted. The results are shown in thetable below (an average value from 3 stocks of each treatment plot, thesmallest to largest values within the parentheses).

TABLE 15 Line 16-hour lighting 12-hour lighting 8-hour lighting W101 08.0 (7-9) 16.3 (15-18) W102 0 6.6 (6-8) 15.3 (13-18) M101 0   0 (0-0)2.0 (0-4) 

As shown in the results, the ability of M101 to form flower buds wasmarkedly low as compared with the wild type line. In order to examine agenetic feature related to this phenotype, the gene sequences of theselines were determined and compared. As a result, the genetic featuresX-1 and X-2 were found only in M101.

INDUSTRIAL APPLICABILITY

The present invention enables the provision of a stevia plant with highyields of leaves and steviol glycosides and can therefore achieveefficient production of steviol glycosides. The present invention canachieve the selection of individuals having a low ability to form flowerbuds before flower bud formation and therefore enables improvement inbreeding efficiency.

1. A stevia plant having a low ability to form flower buds as comparedwith the wild type.
 2. The plant according to claim 1, wherein the plantis heterozygous or homozygous for the allele wherein the base at theposition corresponding to position 90 of SEQ ID NO: 150 is G, and/or isheterozygous or homozygous for the allele wherein the base at theposition corresponding to position 108 of SEQ ID NO: 150 is G.
 3. Theplant according to claim 1 or 2, further having at least one of thefollowing genetic features (1) to (7). (1) Homozygous for the allelewherein the base at the position corresponding to position 40 of SEQ IDNO: 2 is T. (2) Homozygous for the allele wherein the base at theposition corresponding to position 44 of SEQ ID NO: 3 is T. (3)Homozygous for the allele wherein the base at the position correspondingto position 41 of SEQ ID NO: 4 is C. (4) Homozygous for the allelewherein the portion corresponding to positions 55-72 of SEQ ID NO: 5 isdeleted. (5) Homozygous for the allele wherein the base at the positioncorresponding to position 201 of SEQ ID NO: 1 is A. (6) Heterozygous forthe allele wherein the base at the position corresponding to position 49of SEQ ID NO: 6 is A. (7) Homozygous for the allele wherein the base atthe position corresponding to position 49 of SEQ ID NO: 6 is A.
 4. Theplant according to claim 3, having at least one of the followingfeatures (1) and (2). (1) Comprising 3% or more of RebD per unit mass ofa dried leaf. (2) Comprising 0.2% or more of RebM per unit mass of adried leaf.
 5. The plant according to any one of claims 1 to 4, whereinthe plant is a non-genetically modified plant.
 6. The plant according toany one of claims 1 to 5, wherein the plant comprises a stevia plantsubjected to a mutagenesis treatment and a progeny plant thereof.
 7. Aseed, a tissue, a tissue culture or a cell of the plant according to anyone of claims 1 to
 6. 8. The tissue, tissue culture or cell according toclaim 7, which is selected from an embryo, a meristem cell, a pollen, aleaf, a root, a root apex, a petal, a protoplast, a leaf section and acallus.
 9. A method of producing a stevia plant having a low ability toform flower buds, the method comprising a step of crossing the plantaccording to any one of claims 1 to 6 with a second stevia plant. 10.The method according to claim 9, wherein the second plant is the plantaccording to any one of claims 1 to
 6. 11. An extract of the plantaccording to any one of claims 1 to 6, or of the seed, tissue, tissueculture or cell according to claim 7 or
 8. 12. A method of producing astevia extract, comprising a step of obtaining an extract from the plantaccording to any one of claims 1 to 6, or from the seed, tissue, tissueculture or cell according to claim 7 or
 8. 13. A method of producing asteviol glycoside purified product, comprising: a step of obtaining anextract from the plant according to any one of claims 1 to 6, or fromthe seed, tissue, tissue culture or cell according to claim 7 or 8; anda step of purifying a steviol glycoside from the obtained extract. 14.The method according to claim 13, wherein the steviol glycosidecomprises rebaudioside A, rebaudioside B, rebaudioside C, rebaudiosideD, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N,rebaudioside 0, stevioside, steviolbioside, rubusoside, dulcoside A or acombination thereof.
 15. A method of producing a food or beverage, asweetener composition, a flavor or a medicament, comprising: a step ofproviding the extract according to claim 11, or a purified productthereof; and a step of adding the extract or the purified product to araw material for the food or beverage, sweetener composition, flavor ormedicament.
 16. A method of screening for a stevia plant with a lowability to form flower buds, comprising a step of detecting from thegenome of a test stevia plant the presence and/or the absence of agenetic feature of being heterozygous or homozygous for the allelewherein the base at the position corresponding to position 90 of SEQ IDNO: 150 is G, and/or the base at the position corresponding to position108 of SEQ ID NO: 150 is G.
 17. The method according to claim 16,further comprising a step of detecting from the genome of a test steviaplant the presence and/or the absence of the following genetic features(1) to (7). (1) Homozygous for the allele wherein the base at theposition corresponding to position 40 of SEQ ID NO: 2 is T. (2)Homozygous for the allele wherein the base at the position correspondingto position 44 of SEQ ID NO: 3 is T. (3) Homozygous for the allelewherein the base at the position corresponding to position 41 of SEQ IDNO: 4 is C. (4) Homozygous for the allele wherein the portioncorresponding to positions 55-72 of SEQ ID NO: 5 is deleted. (5)Homozygous for the allele wherein the base at the position correspondingto position 201 of SEQ ID NO: 1 is A. (6) Heterozygous for the allelewherein the base at the position corresponding to position 49 of SEQ IDNO: 6 is A from the genome of the test stevia plant. (7) Homozygous forthe allele wherein the base at the position corresponding to position 49of SEQ ID NO: 6 is A from the genome of the test stevia plant.
 18. Themethod according to claim 16 or 17, wherein the step of detecting agenetic feature is performed by use of CAPS method, dCAPS method orTaqMan PCR method.
 19. The method according to any one of claims 16 to18, further comprising a step of evaluating the ability to form flowerbuds in a test stevia plant tissue.
 20. A screening kit for a steviaplant with a low ability to form flower buds, comprising a reagent fordetecting the presence and/or the absence of a genetic feature of beingheterozygous or homozygous for the allele wherein the base at theposition corresponding to position 90 of SEQ ID NO: 150 is G, and/orbeing heterozygous or homozygous for the allele wherein the base at theposition corresponding to position 108 of SEQ ID NO: 150 is G.
 21. Thekit according to claim 20, further comprising a reagent for detectingthe presence and/or the absence of the following genetic features (1) to(7). (1) Homozygous for the allele wherein the base at the positioncorresponding to position 40 of SEQ ID NO: 2 is T. (2) Homozygous forthe allele wherein the base at the position corresponding to position 44of SEQ ID NO: 3 is T. (3) Homozygous for the allele wherein the base atthe position corresponding to position 41 of SEQ ID NO: 4 is C. (4)Homozygous for the allele wherein the portion corresponding to positions55-72 of SEQ ID NO: 5 is deleted. (5) Homozygous for the allele whereinthe base at the position corresponding to position 201 of SEQ ID NO: 1is A. (6) Heterozygous for the allele wherein the base at the positioncorresponding to position 49 of SEQ ID NO: 6 is A. (7) Homozygous forthe allele wherein the base at the position corresponding to position 49of SEQ ID NO: 6 is A.
 22. The kit according to claim 20 or 21, whereinthe reagent comprises a primer and/or a probe for use in CAPS method,dCAPS method or TaqMan PCR method.
 23. A method of producing a steviaplant with a low ability to form flower buds, comprising a step ofintroducing a variation from A to G to a position corresponding toposition 90 of SEQ ID NO: 150, and/or a step of introducing a variationfrom T to G to a position corresponding to position 108 of SEQ ID NO:150.
 24. The method according to claim 23, wherein the introduction ofthe variation is performed by a mutagenesis treatment.